Compositions and methods of making brittle-matrix particles through blister pack freezing

ABSTRACT

The present invention includes compositions and methods for treating and delivering medicinal formulations using an inhaler. The composition includes a space filled flocculated suspension having one or more flocculated particles of one or more active agents and a hydrofluoroalkane propellant. A portion of the one or more flocculated particles is templated by the formation of hydrofluoroalkane droplets upon atomization and the templated floc compacts upon the evaporation of the hydrofluoroalkane propellant to form a porous particle for deep lung delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part and claims priority based onU.S. patent application Ser. No. 12/371,573, filed Feb. 13, 2009, whichclaims priority to U.S. Provisional Application Ser. No. 61/028,218,filed Feb. 13, 2008, the contents of each of which are incorporated byreference herein in their entireties.

STATEMENT OF FEDERALLY FUNDED RESEARCH

This invention was made with U.S. Government support under Contract No.CHE987664 awarded by the NSF. The government has certain rights in thisinvention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to readily scalablepharmaceutical manufacturing process to create multiple blister doses ofbrittle-matrix particles for inhalation.

BACKGROUND OF THE INVENTION

Without limiting the scope of the invention, its background is describedin connection with medicinal formulations and compositions for use inpressurized metered dose inhalers. Current methods of delivery haveproduced few examples of suspensions with 1-5% (w/w) mass loadings inHFAs that are stable against settling on time scales of over 60 seconds.As the mass loading increases up to and above 5% (w/w), particles oftenaggregate within aerosolized droplets leading to substantial increasesin d_(a) and thus reduction in fine particle fraction (FPF).

For example, U.S. Pat. No. 6,585,957 relates to medicinal aerosolformulations. The formulation includes a protein or peptide medicament,a fluid carrier for containing said medicament; and a stabilizerselected from an amino acid, a derivative thereof or a mixture of theforegoing. Similarly, U.S. Pat. No. 6,655,381 relates to pre-metereddose magazine for breath-actuated dry powder inhaler. More specifically,a pre-metered dose assembly for consistently supplying precise doses ofmedicament is taught for a breath-actuated dry powder inhaler. Thebreath-actuated dry powder inhaler including the pre-metered doseassembly in combination with a de-agglomerator for breaking upaggregates and micronizing particles of dry powder prior to inhalationof the powder by a patient.

U.S. Pat. No. 7,011,818 relates to carrier particles for use in drypowder inhalers. The powder includes additive material on the surfacesof the carrier particles to promote the release of the active particlesfrom the carrier particles on actuation of the inhaler. The powder issuch that the active particles are not liable to be release from thecarrier particles before actuation of the inhaler. The inclusion ofadditive material (4) in the powder has been found to give an increasedrespirable fraction of the active material

The general method of delivery of drugs to the lungs for the treatmentof numerous pulmonary disorders is through inhalation of the drugparticles. The drug particles are generally in the form of an aerosol ofrespirable sized particles incorporated into a colloidal dispersioncontaining either a propellant, as a pressurized metered dose inhaler(pMDI) or air such as is the case with a dry powder inhaler (DPI).

It is of the upmost importance in the aerosol formulation that thecomposition is stable and the dose discharged from the metered dosevalve is reproducible; however, there are numerous factors thatinfluence these features, e.g., creaming, or settling, after agitationare common sources of dose irreproducibility in suspension formulations.Another concern is the flocculation of the composition after agitation.This flocculation often results in dose irreproducibility and as such,it is an undesirable process and composition and is often seen inaerosol formulations containing only medicament and propellant orformulation contains small amounts of surfactants. Surfactants are oftenincluded in the formulations to serve as suspending aids to stabilizethe suspension or lubricants to reduce valve sticking which also causesdose irreproducibility.

In addition, the drug absorption into the subject from the airwaydependents on numerous factors, e.g., the composition of theformulation, type of solute, the method of drug delivery, and the siteof deposition. Therefore, formulation and device characteristics have adramatic impact upon the rate and extent of peptide absorption from thelung. Dry powder presentations of peptide and protein drugs possessunique opportunities in formulations, which do not occur in liquidpresentations such as pMDIs and nebulized solutions.

One method commonly used to prepare medicament particles for drugformulations into fine powder is spray drying. Spray drying formsspherical particles that are often hollow thus resulting in a powderwith low bulk density compared to the initial material, othercharacteristics include particle size distribution, bulk density,porosity, moisture content, dispersibility, etc. In addition, the spraydried particles demonstrate poor flow characteristics. The spray dryingprocess requires heating of the formulation making it drying lessdesirable for heat sensitive compounds such as peptide and proteindrugs. For these reasons spray dried particles often suffer fromadhesion and poor flowability to the extent that dose accuracy becomes aproblem.

SUMMARY OF THE INVENTION

The present invention provides for the dispensing of poorly watersoluble compositions and/or protein via pMDI. As stated previously,sub-micron particles are desirable for drug delivery because smallerparticles provide a larger surface area/mass ratio for dissolution.Milling is a common particle size reduction method; however, the millingprocess has been shown to produce partially amorphous drug domains.Although amorphous particles may be desirable for certain applications(e.g., to raise solubility for enhanced bioavailability), they areequally undesirable in many applications (e.g., the drug nanoparticlesmay crystallize upon storage). Thus the inventors recognized that it isimportant to find ways to make crystalline nanocrystals without the needto use milling.

The present invention provides for the formation of stable suspensionsof very low density flocs of rod-shaped drugs in hydrofluoroalkanepropellants for pressurized meter dose inhalers (pMDI) and fortemplating the flocs to achieve high fine particle fractions inpulmonary delivery.

The present invention also provides a unit-dose delivery system used asa template for use in a dry powder inhaler. The invention includes aunit-dose delivery system comprising one or more concave indentations; acover positioned to sealed the one or more concave indentations; and abrittle matrix medicinal formulation appropriate for pulmonary deliveryin at least one of the one or more concave indentations, wherein thebrittle matrix medicinal formulation comprises a non-tightly packedporous flocculated web matrix comprising one or more brittle-matrixparticles of one or more active agents, wherein a portion of the one ormore brittle-matrix particles is delivered and templated by theformation of one or more particles upon atomization from the unit-dosedelivery system using a dry powder inhaler to form a respirable porousparticle for deep lung delivery.

The present invention includes a medicinal formulation for use in a drypowder inhaler having a non-tightly packed porous flocculated webcomposition comprising one or more brittle-matrix particles of one ormore active agents, wherein a portion of the one or more brittle-matrixparticles is templated by a patient and/or device induced shearingenergy to form a porous particle for deep lung delivery

The present invention also provides method of making a dispersiblebrittle templated composition for a dry powder inhaler system by coolinga unit-dose delivery system intended for one or more metered doses forinhalation; depositing one or more drops of a drug solution on theunit-dose delivery system, wherein the drug solution comprises one ormore active pharmaceutical ingredients, one or more solvents, and one ormore excipients, where said drop freezes upon contact with the packagingmaterial; lyophilizing the pharmaceutical product to produce anon-tightly packed brittle matrix; equilibrating the non-tightly packedbrittle matrix to room temperature; and combining the non-tightly packedbrittle matrix with a suitable dry powder inhalation device.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIGS. 1A-1D are SEM images of URF particles from surfactant freeformulations;

FIG. 2 is a table of URF Itz powder dispersed in HPFP;

FIGS. 3A and 3B are SEM images of pMDI formulation, while 3C and 3D arethe corresponding graphs of particle size;

FIGS. 4A-4B are TEM images of URF Itz aerosol from pMDI;

FIGS. 5A and 5C are data for the 100% Itz URF samples shown in the SEMimages FIGS. 5B and 5D;

FIG. 5E is a XRD of URF Itz powder;

FIG. 6 is a dissolution graph of particles emitted by pMDI;

FIGS. 7A-7B are SEM images of Charleston sample Dow amorphous Itz;

FIGS. 8A-8C are SEM images of Charleston sample Dow amorphous Itz frompMDI;

FIGS. 9A-9B are graphs and 9C is a SEM characterizing the Itz samplemade by CP;

FIGS. 10A-10C are SEM images of Itz made by CP from pMDI;

FIG. 11 is a table comparing Itz formulations;

FIG. 12 is a table comparing particle dimensions of ACI;

FIGS. 13A-13C are SEM images of milled Itz particles;

FIG. 14 is a graph of the milled control particles;

FIGS. 15A-15D are SEM images of milled aerosolized milled particles;

FIG. 16 is a XRD of milled Itz particles;

FIGS. 17A-17C are SEM images of TFF particles in HPFP;

FIGS. 18A-18D are SEM images of CP Itz particles in HPFP;

FIGS. 19A-19D are SEM images of Dow amorphous in HPFP;

FIGS. 20A-20C are SEM images of milled Itz particles in HPFP;

FIGS. 21A-21C are SEM images of milled Itz particles in HPFP;

FIG. 22 is a table comparing Itz formulations;

FIG. 23 is a table comparing aerosolized particle dimensions of ACI;

FIG. 24 is a graph of the HFA droplet diameter;

FIG. 25 is an illustration of the calculation of Df;

FIG. 26 is an illustration of the calculation of the settling velocitiesof flocs;

FIG. 27 is a SEM image of TFF particles in HPFP;

FIG. 28 is a SEM image of CP Itz particles in HPFP;

FIG. 29 is a SEM image of DOW amorphous in HPFP;

FIG. 30 is a SEM image of milled Itz in HPFP;

FIG. 31 is a SEM image of milled Itz in HPFP;

FIGS. 32A-32C are schematics of particle suspension of hollow sphereparticles (32A), milled or sprayed particles (32B) and TFF rod particles(32C);

FIGS. 33A-33B are images of TFF particle after lyophilization (33A) andafter drying with acetonitrile (33B);

FIG. 34A is an SEM image of BSA particles, FIG. 34B is an SEM image ofBSA:Trehalose, FIG. 34C is an SEM image of milled BSA particles, FIG.34D is an SEM image of spray dried BSA particles, and FIG. 34E is an SEMimage of TFF particles drying with acetonitrile;

FIG. 35 is a graph of the particle sizes measured by static lightscattering for BSA spheres formed by milling and spray drying and BSAnanorods formed by thin film freezing (TFF) suspended in acetonitrilewhere closed symbols indicate sonicated powder and open circles indicateunsonicated powder;

FIGS. 36A-36F are images of suspensions in HFA 227 of TFF particles atφv=0.0077 (FIG. 36A), φv=0.00077 (FIG. 36B), milled particles 5 minutesafter shaking (FIG. 36C) and spray dried particles at 2 minutes aftershaking (FIG. 36D) at φv=0.0077, TFF particles in acetonitrile atφv=0.0077 immediately after shaking (FIG. 36E) and 3 days after shaking(FIG. 36F);

FIGS. 37A-37F are optical microscopy images of BSA particles suspendedin HPFP with TFF particles magnified 4× (FIG. 37A), 10× (FIG. 37B), and60× (FIG. 37C), spray dried BSA particles after 30 seconds at 10× (FIG.37D), after 60 seconds (FIG. 37E), and milled BSA particles after 30seconds at 10× (FIG. 37F);

FIG. 38 is a graph of the particle sizes measured by static lightscattering for BSA nanorods from thin film freezing (TFF) suspended inHFA 227 or HPFP where closed symbols indicate sonicated powder and opencircles indicate unsonicated powder;

FIG. 39A is an optical image of TFF particles after HFA 227 evaporationand FIG. 39B is an SEM image of TFF particles after sonication and HFA227 evaporation;

FIG. 40 is a DLS graph of TFF particles actuated through the pMDI valvesubmerged beneath acetonitrile;

FIG. 41A is a graph of the ACI mass deposition profiles for device (D)and spacer and throat (S+T) and stages 0-7 and FIG. 41B is a graph ofthe APS mass distribution with a formulations on bar charts include BSA(diagonal lines), BSA+Tween 20 (horizontal lines), and BSA:Trehalose1:1+Tween 20 (dotted);

FIGS. 42A-42D are SEM images of BSA aerosol collected from stage 3 ofAndersen cascade impactor for BSA (FIGS. 42A and 42B) and BSA:Trehalose1:1 (FIGS. 42C and 42D);

FIG. 43 is a table of the dosage and aerodynamic properties of TFF,milled, and spray dried particle suspensions in HFA 227;

FIG. 44 is a table of the aerodynamic particle sizes determined by ACIand APS and geometric particle sizes determined by laser diffraction andSEM;

FIG. 45 is a table of the calculation of the van der Waals (VdW)interaction potential Φvdw of BSA particles in HFA 227;

FIG. 46 is a table of the settling behavior of BSA particles prepared byTFF, milling, and spray drying and calculations for porous shellparticles prepared by spray drying, with the aValue determined from theequivalent volume of a sphere measured from laser light scattering; bThedensity difference was determined by ρf−ρL with ρp=1.5 g/cm3;cDetermined from dimensions given by Dellamary et al.; dCalculated forprimary particle with 100 nm thick shell;

FIG. 47 is an optical image of protein pMDI formulations (Lys in HFA 227with a drug loading of 20 mg/mL, Lys in HFA 134a with a drug loading of40 mg/mL, 50 mg/mL, 90 mg/mL, and BSA (BSA) in HFA 227 with a drugloading of 50 mg/mL, left to right) 4 hours after shaking;

FIG. 48 is a SEM micrographs of aerosolized Lys particles (Lys in HFA134a pMDI loaded at 50 mg/mL). Aerosolized particles have geometricdiameters between 8-10 μm (A) and exhibit porous morphology (B) and (C);

FIGS. 49A-49C are a photograph of 110 mg TFF ITZ powder loaded into aglass vial;

FIG. 50 is a graph of the X-ray diffraction (XRD) pattern of ITZ beforeand after exposure to HFA 227;

FIG. 51 is a graph of the Modulated differential scanning calorimetry(mDSC) of TFF ITZ powders before and after exposure to HFA 227 and HPFPand pure ITZ;

FIG. 52 is a graph of the dissolution profile of TFF ITZ particles afterexposure to HFA 227 conducted in pH 7.4 phosphate buffer (0.02% w/vSDS);

FIGS. 53A, 53B and 53C are scanning electron microscopy images of TFFITZ (FIG. 53A) before and (FIG. 53B) after exposure to HFA 227 and (FIG.53C) SEM image of TFF ITZ after pMDI was actuated into water, withoutany exposure to air;

FIG. 54 is a graph of the dynamic light scattering (DLS) measurements ofHFA-exposed TFF ITZ in water;

FIGS. 55A and 55B are scanning electron microscopy images of aerosolizedTFF ITZ (FIG. 55A) and aerosolized TFF ITZ in dissolution media at 37°C. after t=1 minute (FIG. 55B) dissolution media comprised phosphatebuffer (pH=7.4) containing 0.2 w/v SDS;

FIG. 56 is the dissolution study graph comparing the dissolutionprofiles of aerosolized TFF ITZ and aerosolized milled ITZ particles(300 nm) studied in phosphate buffer (pH=7.4) containing 0.2 w/v SDS at37° C.;

FIG. 57 is a graph of the aerodynamic diameters of milled, TFF, and CPdrug compositions measured by the APS 3321/3343 and theAerosizer/Aerodisperser systems;

FIG. 58 is a graph of the aerodynamic particle size distribution for theTFF lys composition;

FIGS. 59A-59C are SEM micrographs of (FIG. 59A) TFF lys nanorods priorto aerosolization and (FIG. 59B) after aerosolization and FIG. 59C is animage at higher magnification of aerosolized TFF lys particles; and

FIG. 60 is a graph of the aerodynamic distribution of brittle-matrixparticles emitted from an ADVAIR DISKUS®.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

The present inventors recognized that the delivery of proteintherapeutics has been largely limited to parenteral delivery due to thechemical and physical instabilities of proteins and challenges inpermeating biological membranes. The present inventors also recognizedthat pulmonary delivery is non-invasive routes offers advantages oflarge alveolar surface area (about 100 m²), rapid absorption across thethin alveolar epithelium (between about 0.1 and about 0.5 μm), avoidanceof first pass metabolism, and sufficient bioavailabilities.

For pulmonary delivery, pressurized meter dose inhalers (pMDI) remainthe most popular delivery device, relative to dry powder inhalers (DPI)and nebulizers, because of low cost, portability, and disposability.Because most drugs, including proteins, are insoluble inhydrofluoroalkane (HFA) propellants, most effort has focused on thedesign of stable suspensions. The lack of understanding of how to formthese stable suspensions has limited the development of viableformulations. Although certain proteins in suspensions may potentiallybe natured by HFAs, the low degree of contact in the solid state withthe solvent, relative to solutions, is highly beneficial in someinstances, e.g., insulin, lysozyme, catalase and rhDNase I.

To achieve high deposition of aerosolized particles in the deep lung,the aerodynamic diameter (d_(a)) should range between about 1-5 mm. Suchprotein particles may be produced by milling, spray drying, and sprayfreeze-drying (SFD). Milling processes can generate significant amountsof heat on localized areas of the protein particle which can lead todenaturation. In spray drying and SFD processes, proteins may adsorb andsubsequently denature and aggregate at the large gas-liquid interfacecreated upon atomization of droplets on the order of about 10-100 mm,although this effect may be mitigated with interfacially activeexcipients. Limited process yields, in terms of weight of protein, forspray drying (about 50-70%) and SFD (about 80%) are a major concern forhighly valuable proteins.

The present inventors recognized that methods and devices currently usedin the art have a significant challenge in producing protein particleswith over about 90% yield, the optimal d_(a) for deep lung delivery, andhigh stability against aggregation. In fact, there have been fewsuspensions in the art that provide a 1-5% (w/w) mass loadings in HFAsand are stable against settling on time scales of over 60 seconds. Asthe mass loading increases up to and above 5% (w/w), particles oftenaggregate within aerosolized droplets leading to substantial increasesin d_(a) and thus reduction in fine particle fraction (FPF).

Flocculation and settling can lead to irreversible particle aggregationas well as variable dosing between actuations. For example, suspensionsof spherical particles formed by milling or spray drying oftenflocculate and settle in less than 60 seconds. Consequently, theefficiency of pMDIs is often limited for suspensions of proteins, aswell as low molecular weight drugs, with typical FPFs between about5-30%. Although surfactants and co-solvents, such as ethanol, couldpotentially stabilize the suspension, the surfactants currently approvedby the FDA for inhalation are insoluble in HFAs. Even for solublesurfactants, the surfactant tails are often not solvated well enough byHFAs, which have low polarizabilities and van der Waals forces, toprovide steric stabilization. Thus, the present inventors have developeda new surfactant structures by achieving a fundamental understanding ofthe molecular interactions with atomic force microscopy and theory. Thepresent inventors have also developed a method to minimize the use ofco-solvents that can chemically destabilize drugs and modify proteinconformation.

An alternative approach is to modify the particle morphology to enhancethe colloidal stability of the primary particles. Large porous particlesor hollow particles with porous or nonporous shells formed by spraydrying were stable against settling for at least about 4 hours whensuspended in HFAs. Respirable fractions were as high as 68%. Here, thepresence of pores filled with HFA decreases the density difference ofthe particle with the surrounding HFA media and reduces van der Waalsattractive forces between particles. Additional reports of settlingrates, primary particle aggregation, and changes in fine particlefraction, especially after storage, will be beneficial for furtherunderstanding this approach. Recently, large porous nanoparticle (LPNP)aggregates, with d_(a) optimized for dry powder inhaler (DPI) pulmonarydelivery, have been formed by spray drying of aqueous suspensions ofsubmicron particles.

Upon contact with lung tissue, these particles break up intonanoparticles to facilitate dissolution and absorption. To extend thisapproach to delivery with a pMDI, each LPNP can be stabilized as anindividual entity in a colloidal dispersion as shown in FIG. 1, if theLPNPs do not aggregate and settle. An alternative approach for efficientnanoparticle delivery to the deep lung is to nebuilize nanoparticledispersions in aqueous media.

Spray freezing into liquids (SFL), and thin film freezing (TFF), havebeen shown to produce high surface area, stable rod-like particles withabout 50-100 nm diameters and high aspect ratios, despite slower coolingrates than in SFD. The stability of lactase dehydrogenase, based onenzymatic activity, was increased in these processes relative to SFD.This increase was achieved by lowering the area of the gas-liquidinterface, which has been shown to denature proteins.

The present invention provides a method of forming suspensions againstsettling stable of BSA particles in HFA 227 without stabilizingsurfactants or co-solvents in order to achieve high fine particlefractions in pMDI delivery. In stark contrast to the methods currentlyused in the prior art, the present invention provides a method ofpurposely flocculate the particles in the HFA to prevent settling (i.e.,the opposite of the prior art). Spheres, produced by milling or spraydrying, were added to HFA 227, but they produced dense flocs thatsettled rapidly. Asymmetric particles, such as rods, may be expected topack less efficiently to form much lower density flocs with greater freevolume than spheres. Rods were produced by TFF.

FIGS. 1A-1D are SEM images of URF particles from surfactant freeformulations. The present invention provides very light open flocs in anHFA that occupy the entire vial and stack upon each other to preventsettling for months, as illustrated in FIG. 1. The morphology wasdetermined by SEM of the original particles and after solvent removal ofparticles suspended and sonicated in acetonitrile or HFA 227. Theflocculation is reversible, in that the flocs break up into submicronprimary rod particles upon transfer to a more polar solventacetonitrile. The particles were also studied in 2H,3H perfluoropentane(HPFP), a non-volatile surrogate for HFA 227, to analyze floc size byoptical microscopy and static light scattering. The d_(a) values weredetermined with an Andersen cascade impactor (ACI) and aerodynamicparticle sizer (APS) and d_(g) values with static light scattering andSEM micrographs. The emitted HFA droplets, on the order of about 25 μm,were utilized to break apart and template the highly open flocs as seenin FIG. 1. Upon evaporation of the HFA, the shrinkage of the flocs fromcapillary forces produces smaller and denser porous particles withdesirable d_(a).

The particle volume fractions and fractal dimensions for flocs composedof either cylindrical (rods) or spherical primary particles have beencharacterized. Calculations of van der Waals energies between suspendedparticles are presented to explain floc formation and break up of thefloc into subdomains upon templating the flocs with the HFA droplets.The particle shrinkage during HFA evaporation leads to the finalaerosolized particle size and porosity as explained with a materialbalance. The present invention provides a novel approach offlocculating, templating, and shrinking the particles results in properd_(a) with low polydispersities without surfactants or co-solvents.Thus, the present invention circumvents the classical paradigm ofattempting to stabilize colloidal dispersions of preformed primaryparticles with surfactants. The flocculation for achieving stablesuspensions and high fine particles fractions without the need forsurfactants of the present invention is of practical interest for wideclasses of low and high molecular weight pharmaceuticals andbiopharmaceuticals that can be formed into nanorods.

Dry powder inhalers may use the flocs of asymmetric particles for dosedelivery. Currently dry powder inhalers do not use flocs of asymmetricparticles with high aspect ratios.

The flocs can break up more easily under the influence of the shearforces in the dry powder inhaler than more dense particles with loweraspect ratios. The break up of the flocs will produce smaller flocscomposed of particles with appropriate aerodynamic diameters for deeplung delivery. Currently, the efficiency of delivery by dry powderinhalers can be limited by the inability of the air to break up theparticles. Furthermore, small high aspect ratio primary particles thatreach the deep lung will have higher dissolution rates, as a consequenceof higher surface areas. Most of the benefits described for therapy withflocs composed of anisotropic particles described in this applicationwill also be present for delivery with dry powder inhalers. The particlemay be loaded into the dry powder inhaler by a variety of methods. Theymay be compacted into blister packs in the solid state. They may also beloaded as colloidal suspensions in a solvent, where the solvent is aliquid, compressed gas, for example a hydrofluoralkane. The evaporationof the solvent may be used to compact the flocs to raise the finalparticle density in the dry powder inhaler. In addition, the flocs maybe formed directly in a component of the dry powder inhale device bythin film freezing. As described above for PMDIs, this approach does notuse particles that are pre-formed to design the aerodynamic diameter ofthe aerosol particle. Instead, the aerodynamic diameter is generated inthe air ways by the shear forces upon rupture of the flocs. Thisaerodynamic diameter is not present in the starting flocs. Thus, thepresent invention circumvents the classical paradigm of attempting todesign the aerodynamic diameters of pre-formed individual particlesprior to loading into the dpi.

Bovine serum albumin (BSA), trehalose, and polyoxyethylene sorbitanmonolaurate (Tween 20) were purchased from Sigma (St. Louis, Mo.). Thepropellant 1,1,1,2,3,3,3-heptafluoroprane (HFA 227) was purchased fromHoechst (Frankfurt, Germany) and 2H,3H-Perfluoropentane (HPFP) waspurchased from SynQuest Labs Inc. (Alachua, Fla.). The Micro BCA ProteinAssay Reagent Kit was obtained from Pierce (Rockford, Ill.). The waterwas deionized by flowing distilled water through a series of 2×7 L mixedbed vessels (Water and Power Technologies, Salt Lake City, Utah)containing 60:40 anionic:cationic resin blends.

BSA powders were prepared by the thin film freezing (TFF) processdescribed previously. Briefly, 5 mg/mL feed solution of BSA in 10 mMpH=7.4 potassium phosphate buffer was passed at a flow rate of 4 mL/minthrough a 17 gauge (e.g., 1.1 mm ID, 1.5 mm OD) stainless steel syringeneedle. The droplets fell from a height of 10 cm above a rotatingstainless steel drum (12 rpm) 17 cm long and 12 cm in diameter. Thehollow stainless steel drum was filled with dry ice to maintain a drumsurface temperature of 223 K. On impact, the droplets deformed into thinfilms and froze. The frozen thin films were removed from the drum by astainless steel blade and transferred to a 400 mL PYREX® beaker filledwith liquid nitrogen. The excess liquid nitrogen was evaporated in a−80° C. freezer.

A Virtis Advantage Lyophilizer (The Virtis Company, Inc., Gardiner,N.Y.) was used to dry the frozen slurries. Primary drying was carriedout at −40° C. for 36 hrs at 300 mTorr and secondary drying at 25° C.for 24 hrs at 100 mTorr. A 12 hour linear ramp of the shelf temperaturefrom −40° C. to +25° C. was used at 100 mTorr.

Spray drying was performed with a Buchi Model 190 mini spray dryer(Brinkmann, Westbury, N.Y.). A 10 mg/mL BSA feed solution in 10 mMpotassium phosphate buffer (pH=7.4) was atomized using a 0.5 mm ID twofluid nozzle with an atomizing air flow rate of 200 mL/s. The liquidprotein formulation was pumped through the nozzle by a peristaltic pump(VWR, Bridgeport, N.J.) at a flow rate of 5 mL/min using 5 mm IDsilicone tubing. The inlet temperature for the heated aspirator air wasset to 150° C. at a flow rate of 1000 L/hr. The resulting outlettemperature from the above conditions was 80° C.

Bulk BSA powder as received was suspended at 5 mg/mL in acetonitrile.The BSA suspension was placed in a mill filled with 50 ceramic ballsapproximately 1 cm in diameter and milled on a mechanical roller for 24hours. The milled BSA suspension was dried in the Virtis AdvantageLyophilizer at a shelf temperature of 30° C. for 12 hours at 1000 mTorr.

Dry powders were placed in 60 mL glass bottles (Qorpak, Bridgeville,Pa.) and pre-cooled in a −80° C. freezer. HFA 227 was also pre-cooled ina −80° C. freezer and poured into the bottles containing the proteinpowders to form 0.7% (w/w) suspensions. The bottles were packed in dryice and the suspensions were then sonicated for 2 minutes using aBranson Sonifier 450 (Branson Ultrasonics Corporation, Danbury, Conn.)with a 102 converter and tip operated in pulse mode at 35 watts.Approximately 5 mL aliquots of the suspension were then dispensed into a500 mL acetonitrile bath for particle size analysis by static lightscattering with a Malvern Mastersizer-S (Malvern Instruments, Ltd.,Worcestershire, UK). Typical obscuration values ranged from about 11 toabout 13%. Next, 10 mL of the cooled protein formulations were dispensedinto 17 mL glass pMDI aerosol vials (SGD, Paris, France) and fitted withmetering valves containing 100 μL metering chambers (DF10 RC 150, Valoisof America, Inc., Congers, N.Y.). The vials were then allowed to warm upto room temperature.

The dried powders were also suspended in acetonitrile at a concentrationof 5 mg/mL and sonicated for about 2-3 minutes in the same mannerdescribed above. Approximately 2 mL of the sonicated suspension wasdispersed into a 500 mL acetonitrile bath and the particle sizes wereanalyzed by static light scattering.

The amount of BSA was measured using the Micro BCA Protein Assayfollowing protocols provided by Pierce (Rockford, Ill.). Each sample wasmeasured in triplicate with relative standard deviations (% RSD) <2%.The absorbance of the solutions was measured at 562 nm in a 96 wellplate spectrophotometer (μQuant Model MQX200; Biotek Instruments Inc.,Winooski, Vt.). Untreated BSA was used to prepare the protein standardsat concentrations between about 2 and 30 μg/mL.

The protein suspensions in HFA were actuated once through the firingadaptor of a dosage unit sample tube (26.6 mm ID×37.7 mm OD×103.2 mmlength; 50 mL volume; Jade Corporation, Huntingdon, Pa.). The firingadaptor was removed, and 40 mL of DI water was added to dissolve theprotein. The sampling tube was shaken and allowed to sit for at least 30min. to assure that the protein was dissolved in water. The proteinconcentration was determined using the Micro BCA protein assay inconjunction with the μQuant spectrophotometer. The glass vial containingthe HFA protein suspension was weighed before and after each actuationto assure that the proper dose had been released. The measurement wasrepeated 3 times to get an average dose delivered through the valve(DDV) for each formulation.

To characterize the aerodynamic properties of the particles, aneight-stage Andersen cascade impactor (ACI) (Thermo-Andersen, Smyrna,Ga.) with an attached 15 cm spacer and an air flow-rate of about 28.3L/min was used to quantify mass median aerodynamic diameter (MMAD),geometric standard deviation (GSD), fine particle fraction (FPF), andemitted dose (ED). Initially 3 shots were sent to waste, and the next 5shots were made into the ACI. The interval between shots was betweenabout 15-30 seconds to prevent cooling of the metering chamber andsubsequent moisture condensation. After the last dose was discharged,the glass vial was removed from the impactor and the valve stem andactuator were rinsed separately with a known volume of DI water. Eachplate of the impactor was placed in a separate container with a knownvolume of DI water and soaked for 30 minutes to assure completedissolution. The protein concentrations were then measured with theMicro BCA Protein Assay.

The d_(a) of the protein particles were also determined in triplicatewith an Aerodynamic Particle Sizer (APS) 3321 (TSI, Shoreview, Minn.).The throat and spacer from the ACI were placed over the inlet of the APSand the airflow rate through the inlet was 5 L/min. Each formulation wasshot once through the spacer and throat. The particle size range by masswas determined with the Aerosol Instrument Manager (AIM) softwareprovided by TSI.

To obtain aerosolized particles for scanning electron microscopy (SEM)(Hitachi Model S-4500, Hitachi Ltd, Tokyo, Japan) analysis, doublecarbon adhesive tape was applied to stage 3 of the ACI. Each formulationwas actuated once through the ACI with an air flow rate of 28.3 L/min.The carbon tape was removed from stage 3 and applied to an aluminum SEMstage, which was transferred rapidly to a Pelco Model 3 sputter-coaterto minimize exposure to moisture. Total exposure to the atmosphere wasless than 1 minute. The SEM micrographs were then characterized withimaging software (Scion, Frederick, Md.) to determine the particle sizedistribution of at least 100 particles.

The aerosolized particles were also characterized by static lightscattering. Each formulation was actuated once through the ACI spacerand throat. The aerosol exited the outlet of the throat downwards 5 cmdirectly above the laser beam of the Malvern Mastersizer S. For eachformulation 100 measurements of the aerosolized spray were made every 5ms. The recorded measurements were then averaged to give the finalprofile of the aerosolized particles on a volume basis.

Moisture contents in the vials of each formulation were tested with anAquatest 8 Karl-Fischer Titrator (Photovolt Instruments, Indianapolis,Ind.) according to the method described by Kim et al. A 19 gauge needlewas inserted through the septum of the titration cell with the needletip placed below the reagent, and each formulation was measured intriplicate. For all formulations tested the moisture content wasapproximately 500 ppm. The pure HFA was found to have a moisture contentof 250 ppm. The total amount of moisture to the amount of proteinparticles was 7% (w/w).

The particles were initially dispersed by pipette mixing in HPFP andwere observed for about 2 minutes with a Nikon OPTIPHOT 2-POL opticalmicroscope with an attached MTI CCD-72× camera (Nikon, Tokyo, Japan).Pictures were taken 30 and 60 seconds after initial dispersion in HPFP.

FIG. 2 is a table of URF ITZ powder dispersed in HPFP. The μQuantspectrophotometer was used to measure turbidity at 350 nm tocharacterize BSA aggregation. Dry powders of BSA were reconstituted to 1mg/mL and 3×300 uL aliquots of each formulation were placed in a 96 wellFalcon plate which was set in the spectrophotometer.

Particles of BSA suspended in acetonitrile were analyzed by acustom-built dynamic light scattering (DLS) apparatus. The scatteringangle was set to 90° and the data were analyzed a digital autocorrelator(Brookhaven BI-9000AT) and a non-negative least-squares (NNLS) routine(Brookhaven 9KDLSW32). The suspension concentration was 0.5 mg/mL whichgave a measured count rate of approximately 150 kcps. Measurements weremade over a period of about 2 minutes.

Approximately 100-300 mg of protein powder was loaded into a 100 mLgraduated cylinder. The tap density of the protein particles wasmeasured with a Vankel tap density meter (Varian, Palo Alto, Calif.).

FIGS. 3A and 3B are SEM images of pMDI formulation, while 3C and 3D arethe corresponding graphs of particle size. The fluffy BSA particles madeby TFF shown in FIG. 2A had a low tap density of 0.0064 g/cm³. Themorphology of the BSA powder prepared by TFF was interconnected rods 50nm in diameter as seen in FIG. 3A. With the addition of 5 mg/mLtrehalose to the BSA feed solution, similar rods were produced, as wellas fine 50-100 nm relatively spherical particles FIG. 3B. Similarmorphologies were observed previously for lysozyme produced by TFF at223 K. The BSA particles prepared by wet milling as seen in FIG. 3C didnot have high external porosity like the TFF particles, but were in theform of cubes with smooth sides with 400-800 nm dimensions. Lastly,spray drying BSA at a feed concentration of 10 mg/mL formed proteinparticle spheres 3-6 μm in diameter with smooth surfaces as seen in FIG.3D.

For characterization by static light scattering, the various BSAparticles suspended in acetonitrile were sonicated for about 2 minutes.FIGS. 4A-4B are TEM images of URF ITZ aerosol from pMDI. As shown inFIG. 4 the d(v,50) values were 330 nm, 410 nm and 6.3 μm for the TFF,milled and spray dried BSA particles, respectively, consistent with thesizes in the SEMs. Thus, the primary particles remain dispersed inacetonitrile and do not aggregate. As demonstrated previously withlysozyme, the cooling rate in the TFF process for BSA was sufficientlyfast to form high surface area powders that redisperse to 330 nmparticles in acetonitrile, with little sonication (less than about 2minutes). As a further indication of high tendency of the nanorods todeaggregate and disperse in acetonitrile, even with no sonication 2peaks were observed with maxima at 330 nm and 20 μm, with approximately50% of the particles by volume below 1 μm as shown in FIG. 4. Thus theaggregation of the nanorods in the powder state is highly reversible.

To compliment the light scattering results by SEM, the sonicatedsuspensions in ACN were frozen by drip freezing into liquid nitrogen.The acetonitrile was then removed by lyophilization leaving fluffyparticles with an approximate tap density of 0.012 g/cm³ (FIG. 2B). Whenthe particles were redispersed in acetonitrile the measured particlesize profile was d(v,50)=330 nm which was similar to the profile in FIG.4 of the original TFF dispersion, indicating that the lyophilizationprocess did not cause irreversible particle aggregation. As observed bySEM, the morphology in FIG. 3E were 50-100 nm diameter rods, similar tothe interconnected rods of the original TFF powder in FIG. 3A, andconsistent with the sizes from light scattering results in FIG. 4. Thusexposure to acetonitrile followed by sonication does not alter themorphology significantly.

FIGS. 5A and 5C are data for the 100% Itz URF samples shown in the SEMimages FIGS. 5B and 5D. FIG. 5E is a XRD of URF ITZ powder. The driedTFF BSA particles were suspended in HFA 227 and acetonitrile (ACN) at0.70% (w/w) corresponding to a volume fraction in the vial φ_(v) of0.0077, as determined from the true density of BSA ρ_(p)=1.3 g/cm³ asshown in FIG. 5. As shown in FIG. 5A, the particles did not settle evenafter 1 year in storage in HFA 227. Immediately upon adding HFA, theparticles formed flocs that filled the entire volume of the vial. For acontrol with an extremely low φ_(v) of only 0.070% (w/w) as shown inFIG. 5B the loose buoyant flocs still filled approximately half the HFAvolume. For the milled BSA nanoparticles, the suspension initiallyappeared to be uniform (as in FIG. 5A), but the particles settled to thebottom after only 5 minutes as shown in FIG. 5C. Since these particlessettled in HFA 227 (1.41 g/cm³), the milling may have compacted theparticles to ρ above 1.3 g/cm³. These particles creamed in HPFP (1.59g/cm³). Thus, it was estimated that ρ_(p)˜1.50 g/cm³, the average of thetwo solvent densities. The spray dried particles dispersed well withshaking, but creamed after only 2 minutes as shown in FIG. 5D. The TFFnanorods suspended in acetonitrile and sonicated for 2 minutes formed amilky uniform dispersion as shown in FIG. 5E. After 3 days the particleshad settled as shown in FIG. 5F. The dispersion/settling behavior shownin FIGS. 5E and 5F was also observed for milled and spray driedparticles in acetonitrile (data not shown) with settling in about 3 daysand about 30 minutes, respectively.

Because the vapor pressure of HFA 227 is above ambient at 25° C. (about500 kPa), the particles were not studied in situ by microscopy or lightscattering. Instead, the particles were studied at ambient pressure inHPFP, a surrogate nonvolatile solvent. Because HPFP has a similarpolarity and polarizability as HFA 227, attractive forces betweensolutes such as budesonide are similar in both solvents on the basis ofatomic force microscopy (AFM). FIG. 6 is a dissolution graph ofparticles emitted by pMDI. According to light microscopy (FIG. 6A), theTFF particles in HPFP were in the form of loosely packed aggregates ofrods as shown in FIG. 6B and FIG. 6C). The particles were in 200-300 μmflocs with subdomains on the order of 25 μm within 5 seconds afterdispersing the particles by pipette mixing (see FIGS. 6A and 6B). Forthe spray dried (as shown in FIGS. 6D and 6E) and milled (as shown inFIG. 6F) particles, 100 μm flocs formed in 30 seconds and grew to over200 μm in 60 seconds.

FIGS. 7A-7B are SEM images of Charleston sample Dow amorphous ITZ. Theseflocs were more densely packed and composed of larger primary particlesthan those formed from TFF particles. These sizes were consistent withstatic light scattering measurements of the sonicated and unsonicatedsuspensions in HPFP with d(v,50) values between about 215-259 μm.

To better anticipate the fate of particles throughout the pMDI deliveryprocess, it would be beneficial to determine how reversibly the nanorodsare bound together in the flocs. The elevated pressure of the HFAcomplicates in situ light scattering. Furthermore, the HFA suspensioncould not be lyophilized to prepare a sample for SEM since the freezingpoint (−131° C.) of HFA 227 is too low to for conventional shelflyophilizers. To investigate the effect of HFA evaporation on theparticles, HFA was cooled to −80° C., well below the boiling point of−16° C., and completely evaporated. The TFF particle residue onlyoccupied approx. 1 mL (tap density of 0.10 g/cm³, FIG. 8A), an order ofmagnitude less than that of the starting TFF bulk powder as shown inFIG. 2A.

FIGS. 8A-8C are SEM images of Charleston sample Dow amorphous ITZ frompMDI. The morphology shown in FIG. 8A was rods with 100 nm diameters(see FIG. 8B), similar to the original TFF particles in FIG. 3A.Therefore, exposure to HFA 227 followed by sonication did notsignificantly alter the microscopic nanorod morphology. However, thedensified aggregates of the nanorods formed by capillary forces uponevaporation as shown in FIG. 5B of HFA were not redispersible in HFA orin ACN. For a sonicated TFF paticle dispersion in ACN, the lyophilizedpowder was redispersible in ACN and in HFA, forming suspensionsidentical to FIG. 5A. Thus it appeared that the capillary forces duringHFA evaporation and perhaps moisture produced irreversible aggration ofthe nanorods.

Given the challenges of in situ high pressure light scattering,lyophilization of HFA 227, and compaction of the TFF rods by capillaryforces upon HFA evaporation, a more practical approach was to transferthe suspension from HFA 227 to a less volatile solvent. If the nanorodsredisperse to primary particles in a good solvent such as acetonitrile,then they were not aggregated irreversibly in HFA 227. A 2 mL aliquot ofthe cold TFF suspension was mixed directly with 500 mL of acetonitrileat 25° C. The flocs deaggregated nearly completely to individual primaryparticles with over 80% of the volume distribution between 100 nm and 1μm, and a maximum at 11 μm as shown in FIG. 7. A relatively small peakwas centered at 5 μm. The distributions nearly matched those of theoriginal TFF particles in ACN. In a complimentary experiment, the valveof the pMDI containing was submerged into acetonitrile and actuated. Aslightly turbid dispersion was formed with an approximate particleconcentration of 0.5 mg/mL, too low for detection by static lightscattering, but not for DLS.

FIGS. 9A-9B are graphs and 9C is a SEM characterizing the ITZ samplemade by CP. From DLS, the particle size was 1-2 μm much smaller than the250 μm floc size in HFA. Therefore, both experiments indicate theloosely connected flocculated nanorods in HFA were reversible and brokeup into primary nanorods, which will be shown to be beneficial for lungdelivery.

Aggregates of protein molecules did not appear to form according tooptical density (OD) measurements at 350 nm of 1 mg/mL BSA [43,61]. TheOD was the same at 0.042 for aqueous solutions in 10 mM phosphate pH=7.4buffer prepared from bulk and TFF powder, both before and after storagein HFA 227 for 1 week. In the glassy state, BSA is less susceptible toaggregation. The total moisture to BSA content was 7% (w/w) for thesuspended BSA particles in HFA 227 as determined by Karl-Fischertitration. Even at particle moisture contents of 8% (w/w), BSA glasstransition temperatures T_(g) range between 80-100° C. Thus thetemperature was well below T_(g), assuming the HFA 227 did notcontribute to plasticization.

The suspension must be stable for consistent dosing with a pMDI, whichis commonly characterized by the dose (mass) delivered through the valve(DDV) as seen in Table 1.

TABLE 1 ACI results for different protein pMDI formulations at differentprotein concentrations. Bovine serum albumin (BSA) and lysozyme (Lys)formulations shown. % Fine Particle DDV Theoretical FPF Done/ActuationED Formulation (μg) DDV (%) (μg) (μg) TFF BSA 915 ± 21 92 47 ± 4.0 318 ±31  695 ± 133 TFF BSA 326 ± 58 83 43 ± 4.2 202 ± 16 690 ± 71 Tween 20TFF BSA:Tre 1:1 452 ± 54 90 38 ± 2.1 132 ± 19 350 ± 56 Tween 20 TFF BSAunsonicated 625 ± 95 63 — — — Milled BSA 295 ± 17 30 — — — Spray DriedBSA 308 ± 38 31 — — —

The concentration was 10 mg/mL or 0.7% (w/w) in each HFA suspension.Therefore, the theoretically delivered dose per actuation would be 1 mgwith the 100 μL valve. For the BSA TFF particles, the DDV values were92% and 63% of the theoretical delivery dose for the sonicated andunsonicated TFF particles, respectively as seen in TABLE 1. For theBSA:Trehalose 1:1 formulation, it was 90%, and the delivered dose was450 μg/actuation as a consequence of the lower amount of BSA loaded intothe vial. For the milled and spray dried suspensions with rapidsettling, the DDV was only 30-31% of the theoretical loading. Here, theformulation was actuated less than 5 seconds after vigorous shakingTherefore, these suspensions were not tested further for aerosolproperties.

FIGS. 10A-10C are SEM images of ITZ made by CP from pMDI. As shown inTable 2 and FIG. 10, the d_(a) determined from the Andersen cascadeimpactor (ACI) and the Aerodynamic Particle Sizer (APS) were in goodagreement and ranged from 3 to 4 μm, within the optimal 1-5 μm range forpulmonary delivery.

TABLE 2 ACI APS d (v, 50) SEM MMAD ACI MMAD APS Particle DiameterParticle Diameter ρ_(g) Formulation (μm) GSD (μm) GSD (μm) (μm) (g/cm³)BSA 3.1 ± 0.1 1.9 ± 0.1 3.2 ± 0.03 1.6 ± 0.01 9.1 ± 0.9 9.4 0.19 BSA 3.6± 0.1 1.9 ± 0.2 — — 9.9 ± 0.8 9.3 — Tween 20 BSA:Tre 1:1 3.2 ± 0.2 1.8 ±0.1 4.0 ± 0.15 1.7 ± 0.01 7.3 ± 0.5 7.4 — Tween 20

As determined by the ACI, the fine particle fraction (FPF) (particlesless than 4.7 μm) was unusually high [32] for an HFA suspension, rangingfrom 38 to 48%, compared to 5 to 30% for typical suspensions [32],producing a fine particle dose/actuation of approximately 300 μg for thefirst two formulations in TABLE 1. The emitted dose (ED) (amount of drugthat exited the actuator) was approximately 70% of the DDV uponactuation (see TABLE 1 and FIG. 10A). The addition of Tween 20 did notaffect any of the properties of the aerosolized TFF powders in TABLE 1significantly or the suspension stability, indicating that it was notneeded as a stabilizer.

The particles were recovered from the ACI for SEM analysis. The peakdrug mass in the ACI was deposited on stages 3 and 4, with d_(a) between2.0-4.7 μm as shown in FIG. 10A. Therefore, particles were collected onstage 3 (d_(a)=3-4 μm).

FIG. 11 is a table comparing ITZ formulations. The particles were porousand composed of rods with diameters less than 500 nm (see FIGS. 11A and11B), similar in morphology to the original nanorods in FIG. 3A. ForBSA:Trehalose 1:1 the fine 50-100 nm primary particles, shown in FIG.3B, changed morphology to include curved plates with features on theorder of more than one micron as shown in FIGS. 11C and 11D.

The SEMs were analyzed by Scion software o determine the volume averagediameter

$\begin{matrix}{D_{Vol} = \frac{\sum d^{4}}{\sum d^{3}}} & (1)\end{matrix}$

where d is the measured diameter of the particle. The D_(vol) for BSAwas approximately 9 μm, while for BSA:Trehalose 1:1 it was slightlysmaller, 7 μm (TABLE 2). The d_(g) of the aerosolized particles werealso measured by static light scattering. An effective refractive indexn_(e) was calculated according to the Bruggeman mixing rule [66] basedon the volume fraction of BSA in the aerosolized particle φ_(g). Fromthe d_(g) and the d_(a) (see Table 2), the particle density ρ_(g) can bedefined by

d _(a) =d _(g)√{square root over (ρ_(g))}  (2)

where ρ_(g)=0.19 g/cm³. The resulting φ_(g)=ρ_(g)/ρ_(p)=0.14. Withn=1.45 and 1.00 for pure BSA and air, respectively, n_(e)=1.1. As shownin TABLE 2 the volume average d(v,50) particle sizes varied by less than1 μm from the values determined from the SEM micrographs. The consistentd_(g) and d_(a), each measured by two techniques, indicate that TFFparticles form large porous particles, and with the optimal size rangefor pulmonary delivery upon aerosolization. When the TFF particles wereactuated above 10 mM phosphate buffer (pH=7.4) the porous particles wereobserved to dissolve in less than 5 seconds. The high surface areafavors rapid dissolution, which could be advantageous for rapiddissolution rates of proteins that have low solubilities in water.

The van der Waals forces between particles play a key role in thedifferences in colloidal stabilities of various types of primaryparticles and the behavior of the flocs in this study, as depicted inthe summary in FIG. 1. According to the Derjaguin-Landau-Verwey-Overbeek(DLVO) theory, particle stability depends on counteracting theattractive van der Waals forces by electrostatic and/or stericrepulsion. If attractive van der Waals (VdW) forces are dominant at allseparation distances, particles flocculate and may then settle.Currently, electrostatic stabilization in HFAs is not well understood,but atomic force microscopy (AFM) measurements indicate thatelectrostatic forces may be negligible compared to attractive VdWforces. The understanding of steric stabilization in HFAs is in itsinfancy. While novel surfactants are being discovered, developed andapproved, alternative mechanisms form the formation of stablesuspensions in HFAs without surfactants would be useful.

The destabilizing van der Waals attractive forces between suspended areweaker for porous particles or hollow particles with thin solid shells.These particles can be stable for hours in HFAs, compared to non-porous1-5 micron particles, which often flocculate and settle rapidly in lessthan 1 minute (see TABLE 2). Dellamary et al. suggested that theincreased suspension stability resulted from a weaker attractive VdWenergy potential Φ_(vdw) between the particles (FIG. 1A), butquantitative calculations were not presented.

As shown in the Appendix the van der Waals energy Φ_(vdw) is directlyproportional to the Hamaker constant A₁₂₁. In order to compare values ofΦ_(vdw) it is necessary to choose a separation distance, D, betweenparticles. TABLE 3 gives the D where Φ_(vdw) becomes equivalent to thethermal energy 3/2 k_(B)T at 298K.

TABLE 3 Separation Distance Particle Particle Hamaker constant (nm) atType diameter 10²¹ A₁₂₁ (J) Φ_(vdw) = 3/2 KgT Spray dried 5.0 14 270Non-porous Spray dried Porous 5.0 3.8 100 Φ = 0.5 Spray dried Hollow 5.014 120 sphere Φ = 0.12 TFF Nanorods 0.33 14 23 TFF Nanorods 0.33 2.6 6.9

An increase in D required to overcome thermal energy indicates strongerattraction between particles. In TABLE 3, the porous particles withφ=0.5 had a calculated A₁₂₁ (Eq. A.3) that was nearly a factor of 4lower than for the non-porous particles. Consequently, D was a factor of3 smaller. The hollow spheres from TEM images were estimated to have 2-5μm diameters and about 100 nm thick shells. Although the A₁₂₁ for thehollow sphere particles with solid shells was the same as for thenon-porous particles, the calculated D was still lower by a factor of 2as a consequence of the differences in the geometries (Eq. A.5).Therefore, the Φ_(vdw) calculations quantify the benefits of weakerattraction for porous particles or for particles with hollow cores. Areduction in Φ_(vdw) or in D to overcome thermal energy can reduce therate of flocculation over orders of magnitude as described by thestability ratio.

Although, the porous or hollow sphere particles can effectively preventflocculation, the particles are still subject to settling by gravity. Ifporous or hollow sphere BSA particles were suspended at φ_(v)=0.0077,the particles would occupy about 10% of the suspension (as shown in

FIG. 1A) and could potentially settle into a dense sediment. As shown inTABLE 4, the calculated settling rate for a single hollow sphereparticle with a solid shell is 6.4×10⁻⁴ mm/s indicating that theparticles would settle a distance of 2 cm in about 9 hours. The settledparticles would then potentially aggregate irreversibly leading todecreased FPFs upon aerosolization.

TABLE 4 d_(p) d^(floc) (ρ_(L) − ρ_(f)) U_(f) U_(p) Particle Type (μm)(μm) (g/cm³) (mm/s) (mm/s) φ_(f) φ^(flocs) φ_(f) D_(f) TFF 0.33^(a) 2500.00022 0.023 2.4 × 10⁻⁵ 0.00077 0.38 0.0020 2.4 Milled 0.41 1000.0080^(b) 0.13 3.7 × 10⁻⁵ 0.0067 0.11 0.073 2.5 Spray Dried 6.3 1000.040 0.80 8.8 × 10⁻³ 0.0077 0.021 0.36 2.6 Spray Dried- 5.0^(c) —0.013^(d) — 6.4 × 10⁻⁴ — — — — Hollow Sphere ^(a)Value determined fromthe equivalent volume of a sphere measured from laser light scattering^(b)The density difference was determined by ρ_(f) − ρ_(L) with ρ_(p) =1.5 g/cm³ ^(c)Determind from dimensions given by Dellamary et al. (1)^(d)Calculated for primary particle with 100 nm thick shell

The concept in this study of stabilizing suspensions with purposelyflocculated rods is based on the space filling properties of the rodsand the flocs. Experimental and theoretical studies indicate that rodscreate extremely low density flocs and thus fill much greater spacecompared to spheres as illustrated in FIGS. 1B and 1C. For spheres, thevolume fraction of primary particles within a floc φ_(f) is related tothe floc diameter d^(floc), primary particle diameter d_(p), and fractaldimension D_(f), which characterizes the floc structure, by

$\begin{matrix}{\varphi_{f} \approx \left( \frac{d^{floc}}{d_{p}} \right)^{D_{f} - 3}} & (3)\end{matrix}$

Philipse et al. modified Eq. 3 to account for the packing physics ofcylindrical rods of length L and diameter D with the result

$\begin{matrix}{\varphi_{f} \approx {\frac{1}{r} \cdot \left( \frac{d^{floc}}{V_{p}^{1/3}} \right)^{D_{f} - 3}}} & (4)\end{matrix}$

where r=L/D is the aspect ratio. The volume of a TFF cylindrical rod,V_(p)=0.019 μm³, was calculated from the equivalent volume of a spherewith particle diameter d(v,50)=0.33 μm, which was measured by staticlight scattering (as shown in FIG. 4A) in acetonitrile. For a rod withvolume V_(p)=π·D²L/4 and D=0.050 μm (as shown in FIG. 3A), L isdetermined as 0.48 μm and thus r=9.6. For r˜10, the predicted φ_(f) inEq. 4 is ˜1 order of magnitude lower than for spherical particles withequivalent d^(floc), D_(f), and where d_(p) for spheres scales as V_(p)^(1/3) for rods.

The density of a floc ρ_(f) and φ_(f) can be determined experimentallyfrom the visually observed floc settling rate, U_(f) , according toStoke's law

$\begin{matrix}{U_{f} = \frac{d^{{floc}^{2}} \cdot \left( {\rho_{f} - \rho_{L}} \right) \cdot g}{18 \cdot \mu}} & (5)\end{matrix}$

where ρ_(L) and μ are the liquid density and viscosity, respectively,and d^(floc)=250 μm for TFF flocs and 100 μm for spray dried and milledflocs. After solving for ρ_(f) in Eq. 5, φ_(f) may be determined by thestraightforward material balance ρ_(f)=ρ_(L)+φ_(f)·(ρ_(p)−ρ_(L)). Asseen in Table 4, φ_(f) for the TFF particles is 1-2 orders of magnitudelower than for the spherical milled and spray dried particles. From Eq.3 and 4 the calculated D_(f) values are in a narrow range from 2.4 to2.6 in each case. Although the milled and TFF particles have nearlyequivalent d^(floc) and D_(f) values relative to the rods (as seen inTable 4), the 1/r scaling in Eq. 5 for rods accounts for the 1 order ofmagnitude decrease in φ_(f), for a given V_(p), which is consistent withtheoretical prediction above.

The one or more anisotropic particles may have an aspect ratio range ofbetween 0.1 and 2.0 or greater, e.g., the aspect ratio may be 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 and so on.

The extremely low φ_(f) means the flocs will fill a huge volume of spacefor a given φ_(v) (as shown in FIG. 1C). The open nanorod flocs with lowφ_(f) filled large amounts of space in HFA and stacked upon each otherlike tumbleweeds to prevent settling. The volume fraction of flocs inthe HFA suspension, φ^(flocs), is given by φ^(flocs)=φ_(v)/φ_(f)(derivation given in Appendix) where φ^(flocs) s determines the spacefilling capability of the flocs. As φ^(flocs) approaches 1 the flocsoccupy the entire volume of HFA (as shown in FIG. 1C). For the diluteφ_(v)=0.00077 suspension (as shown in FIG. 5B), the calculated φ^(flocs)was about 0.38 (see Table 4) in good agreement with FIG. 5B. At aloading 10 fold higher, φ_(v)=0.0077, the entire vial was white withoutthe appearance of spaces between flocs (as shown in FIG. 5A), asexpected from the low φ_(f) . Here it was not possible to observe asettling rate as the visual appearance did not change for 1 year, themaximum time tested, as the φ^(flocs) of essentially unity preventedsettling. In order for the spherical particles to produce φ^(flocs)=1the required mass loadings for the milled and spray dried particleswould be 6.7% (w/w) and 33% (w/w), respectively, compared to <0.7% (w/w)for the TFF rods.

In contrast to the TFF rods, the hollow sphere particles would settlethe length of the vial (about 2 cm) by gravity in about 9 hoursaccording to Stoke's law for a particle diameter of 5 μm and shellthickness of 100 μm. In the settled state with a high particle volumefraction and contact between protein chains they are more likely to formirreversible particle aggregates by interparticle diffusion andsintering.

The open flocs in HFA 227, that gave the stable suspensions, may beshown to be favored by the relatively strong attractive forces betweenthe primary particles. At first, this may seem counterintuitive to thenormal goal of lowering attractive forces to stabilize colloidaldispersions. Upon addition of the HFA, the relatively strong attractiveforces between the primary rods, Φ_(vdw), cause sticky collisions to“lock in” the open structure rapidly to inhibit collapse of the flocs.For weaker attractive forces between primary particles, collapse hasbeen shown to be more prevalent as particles sample a greater number ofenergetically favorable locations to reduce the interfacial surfacearea. Therefore, rapid flocculation from sticky collisions facilitatesthe formation of low density flocs that fill the entire vial and preventsettling.

In contrast to the flocs in HFA 227, colloidal dispersions of primaryTFF rods in acetonitrile settled in 3 days (as shown in FIG. 5F). Thissettling rate agreed with the predicted settling rate of individualeffective spheres with a diameter of 330 nm from light scattering givenin Table 4. From Table 3, the calculated A₁₂₁ values for BSA inacetonitrile are 1 order of magnitude lower than in HFA 227. Therefore,the stronger attractive forces between particles in HFA relative to ACN,favors formation of open flocs, resulting in more stable suspensionsagainst settling.

Although the 250 μm flocs form stable suspensions, they are too large toproduce optimal d_(a). The shear forces in the actuator are needed tobreak apart the flocs. The calculation of these shear forces is rarelyreported because the turbulence from the immediate onset of HFAevaporation produces complex cavitation events. According to empiricalmodels, aerosolized HFA droplets are typically about 10-30 μm indiameter. Thus we choose an HFA droplet diameter of 25 μm. The shearforces acting on the flocs are sufficiently strong to overcome theattractive van der Waals interactions between primary particles within afloc such that the HFA droplets may template the 250 μm flocs into 25 μmsubdomains with the same φ_(f)=0.0020 as illustrated schematically inFIG. 1. From the high φ^(flocs) (TABLE 1C) it is expected that most ofthe HFA droplets are likely to be filled with a subdomain.

Since direct comparison of calculated shear forces to van der Waalsforces of primary particles within a floc is unfeasible, the concept oftemplating of the 25 μm subdomains is instead supported by a materialbalance on the protein between the volume of the HFA droplet, V_(IIFA),and the volume of the dry aerosolized particle, V_(g), (as shown in FIG.1C) given by

V _(g)·ρ_(g) =V _(HFA)·ρ_(HFA)   (6)

where BSA concentrations are given by ρ_(HFA)=φ_(v) ρ_(p), andρ_(g)=φ_(g)·ρ_(p). It is assumed that the volume fraction of particlesin HFA droplet is approximately equal to φ_(v) as a result of the breakup of the flocs. From the d_(g) and d_(a) in Table 2 and ρ_(g)=0.19g/cm³ (Eq. 2), φ_(g)=0.14. The φ_(g) is nearly 20 times greater thanφ_(v) in the vial. Therefore, the capillary forces in the shrinking HFAdroplets during evaporation collapse the flocs. Eq. 6 is refined torelate φ_(g) to φ_(v) as

$\begin{matrix}{{\varphi_{g} \cdot d_{g}^{3}} = {\frac{f_{BSA}}{f_{HFA}} \cdot \varphi_{v} \cdot d_{HFA}^{3}}} & (7)\end{matrix}$

where d is a diameter, f_(BSA)=0.7 accounts for the mass fraction ofdrug that is emitted from the actuator, and f_(HFA)=0.5 accounts for themass fraction of HFA that exits the actuator orifice to form aerosolizedliquid droplets (relative to vapor).

From Eq. 7 with d_(g)=9.3 μm (TABLE 2), d_(HFA)=25 μm, and φ_(v)=0.0077,the calculated φ_(g)=0.21, which compares reasonably well to theexperimentally determined φ_(g)=0.14. Also the polydispersity in theaerodynamic properties was small. It would be unlikely that any otherfactor besides templating of the flocs with relatively uniform HFAdroplets could explain these low polydispersities.

The control in FIG. 8A supports this argument since the TFF particlesremained below the meniscus of the evaporating HFA 227. The tap densityof the particles was approximately 0.10 g/cm³ (FIG. 8A) which is withina factor of 2 of the calculated density (0.19 g/cm³) of the aerosolizedparticle. Therefore, the capillary forces acting on the TFF particlesduring HFA evaporation compacted the particles into denser aggregateswith a highly desirable value of the d_(a). If needed, the d_(a) may bemanipulated further by varying the valve volume and geometry and the HFAdroplet generation. If the particles had not collapsed partially, theywould have been too large and light for pulmonary delivery. Even afterthis collapse, the porosity and surface area were still relatively highand favorable for high dissolution rates of small molecules and proteinswith limited solubilities, relative to nonporous particles.

High (e.g., about 38-48%) fine particle fractions in HFA 227 pMDIdelivery were achieved with flocculated BSA nanorods stable againstsettling for up to 1 year, without the use of surfactants andcosolvents. Analysis of experimental settling rates of dilutesuspensions indicated that the volume fraction, φ_(f), of the nanorodsin the flocs was an order of magnitude lower than for flocs of sphericalparticles produced by milling or spray drying. The rapid and stickyattractive collisions of nanorods, facilitates the formation of lowdensity flocs (250 μm) which stack upon each other to fill the entiresolvent volume to prevent settling. In contrast, denser flocs ofspherical particles filled much less space and rapidly settled within 60seconds. The novel concept of purposely flocculating nanorods to preventsettling is fundamentally opposite the conventional approach ofstabilizing colloidal dispersions of primary particles. Thereversibility of the nanorod flocs in HFA 227 was demonstrated by breakup of the flocs into individual 330 nm primary rod particles upontransfer to the more polar solvent acetonitrile.

A material balance on a shrinking HFA droplet containing a 25 μm flocsubdomain predicts a final volume fraction of BSA in the aerosolizedparticle in agreement with experiment. Therefore, the attractive van derWaals interactions between primary particles within the floc aresufficiently weak such that the atomized HFA droplets initially templatethe 250 μm flocs into 25 μm subdomains. The aerosolized particles with ad_(a) of 3-4 μm and d_(g) of about 10 μm are optimal for high fineparticle fractions via a pMDI. The concept of forming open flocscomposed of nanorods, that are stable against settling withoutsurfactants, and templating the flocs to achieve optimal d_(a)s and highFPFs is of practical interest for wide classes of low and high molecularweight pharmaceuticals and biopharmaceuticals.

FIG. 12 is a table comparing particle dimensions of ACI. FIGS. 13A-13Care SEM images of milled ITZ particles. FIG. 14 is a graph of the milledcontrol particles. FIGS. 15A-15D are SEM images of milled aerosolizedmilled particles. FIG. 16 is a XRD of milled ITZ particles. FIGS.17A-17C are SEM images of TFF particles in HPFP. FIGS. 18A-18D are SEMimages of CP ITZ particles in HPFP. FIGS. 19A-19D are SEM images of Dowamorphous in HPFP. FIGS. 20A-20C are SEM images of milled ITZ particlesin HPFP. FIGS. 21A-21C are SEM images of milled Itz particles in HPFP.

FIG. 22 is a table comparing ITZ formulations. FIG. 23 is a tablecomparing aerosolized particle dimensions of ACI. FIG. 24 is a graph ofthe HFA droplet diameter. FIG. 25 is an illustration of the calculationof D_(f). FIG. 26 is an illustration of the calculation of the settlingvelocities of flocs. FIG. 27 is a SEM image of TFF particles in HPFP.FIG. 28 is a SEM image of CP ITZ particles in HPFP. FIG. 29 is a SEMimage of DOW amorphous in HPFP. FIG. 30 is a SEM image of milled ITZ inHPFP. FIG. 31 is a SEM image of milled ITZ in HPFP. FIGS. 32A-32C areschematics of particle suspension of hollow sphere particles (FIG. 32A),milled or sprayed particles (FIG. 32B) and TFF rod particles (FIG. 32C).

FIGS. 33A-33B are images of TFF particle after lyophilization (33A) andafter drying with acetonitrile (FIG. 33B). FIG. 34A is an SEM image ofBSA particles, FIG. 34B is an SEM image of BSA:Trehalose, FIG. 34C is anSEM image of milled BSA particles, FIG. 34D is an SEM image of spraydried BSA particles, and FIG. 34E is an SEM image of TFF particlesdrying with acetonitrile. FIG. 35 is a graph of the particle sizesmeasured by static light scattering for BSA spheres formed by millingand spray drying and BSA nanorods formed by thin film freezing (TFF)suspended in acetonitrile where closed symbols indicate sonicated powderand open circles indicate unsonicated powder. FIG. 36A-36F are images ofsuspensions in HFA 227 of TFF particles at φ_(v)=0.0077 (FIG. 36A),φ_(v)=0.00077 (FIG. 36B), milled particles 5 minutes after shaking (FIG.36C) and spray dried particles at 2 minutes after shaking (FIG. 36D) atφ_(v)=0.0077, TFF particles in acetonitrile at φ_(v)=0.0077 immediatelyafter shaking (FIG. 36E) and 3 days after shaking (FIG. 36F).

FIG. 37A-37F are optical microscopy images of BSA particles suspended inHPFP with TFF particles magnified 4× (FIG. 37A), 10× (FIG. 37B), and 60×(FIG. 37C), spray dried BSA particles after 30 seconds at 10× (FIG.37D), after 60 seconds (FIG. 37E), and milled BSA particles after 30seconds at 10× (FIG. 37F). FIG. 38 is a graph of the particle sizesmeasured by static light scattering for BSA nanorods from thin filmfreezing (TFF) suspended in HFA 227 or HPFP where closed symbolsindicate sonicated powder and open circles indicate unsonicated powder.FIG. 39A is an optical image of TFF particles after HFA 227 evaporationand FIG. 39B is an SEM image of TFF particles after sonication and HFA227 evaporation. FIG. 40 is a DLS graph of TFF particles actuatedthrough the pMDI valve submerged beneath acetonitrile. FIG. 41A is agraph of the ACI mass deposition profiles for device (D) and spacer andthroat (S+T) and stages 0-7 and FIG. 41B is a graph of the APS massdistribution with a formulations on bar charts include BSA (diagonallines), BSA+Tween 20 (horizontal lines), and BSA:Trehalose 1:1 +Tween 20(dotted).

FIG. 42A-42D are SEM images of BSA aerosol collected from stage 3 ofAndersen cascade impactor for BSA (FIG. 42A and 42B) and BSA:Trehalose1:1 (FIGS. 42C and 42D). FIG. 43 is a table of the dosage andaerodynamic properties of TFF, milled, and spray dried particlesuspensions in HFA 227. FIG. 44 is a table of the aerodynamic particlesizes determined by ACI and APS and geometric particle sizes determinedby laser diffraction and SEM. FIG. 45 is a table of the calculation ofthe van der Waals (VdW) interaction potential Φ_(vdw) of BSA particlesin HFA 227.

FIG. 46 is a table of the settling behavior of BSA particles prepared byTFF, milling, and spray drying and calculations for porous shellparticles prepared by spray drying, with the ^(a)Value determined fromthe equivalent volume of a sphere measured from laser light scattering;^(b)The density difference was determined by ρf−ρL with ρp=1.5 g/cm³;^(c)Determined from dimensions given by Dellamary et al.; ^(d)Calculatedfor primary particle with 100 nm thick shell. FIG. 47 is an opticalimage of protein pMDI formulations (Lys in HFA 227 with a drug loadingof 20 mg/mL, Lys in HFA 134a with a drug loading of 40 mg/mL, 50 mg/mL,90 mg/mL, and BSA (BSA) in HFA 227 with a drug loading of 50 mg/mL, leftto right) 4 hours after shaking FIG. 48 is a SEM micrograph ofaerosolized Lys particles (Lys in HFA 134a pMDI loaded at 50 mg/mL).Aerosolized particles have geometric diameters between 8-10 μm (A) andexhibit porous morphology (B) and (C).

This invention is a new composition of matter by process for producinghighly concentrated (about 10-90 mg/mL), suspensions of drugs inpressurized metered dose inhalers (pMDIs). This approach may be used formany types of low molecular weight drugs, and for high molecular weightdrugs including peptides and proteins. Dry powders of submicron proteinparticles produced by thin film freezing, a powder formation processdescribed in manuscripts by Overhoff et. al and Engstrom et. al.(incorporated herein), readily disperse when added to ahydrofluoroalkane propellant to form a stable suspension.

Upon actuation, the submicron protein particles contained within thepropellant droplets aggregate to form a porous protein structure (e.g.,8-10 μm) ideal for pulmonary deposition. Pulmonary delivery of proteinsis of great interest because the lungs are far more permeable tomacromolecules compared to other routes into the body, such as thegastrointestinal (GI) tract, and less invasive than parenteral routes.Furthermore, lung concentrations of metabolizing enzymes are lower thanthat found in the GI tract and liver.

At concentrations in an HFA of 10 mg/mL achieved emitted and respirabledoses of 700 μg and 300 μg per actuation, respectively, of bovine serumalbumin (BSA). The new work extends this concept to concentrations of upto 90 mg/mL in HFA 134a. This leads to emitted doses as high as 4mg/actuation as described below in TABLE 5. One of the primarycriticisms levied against pMDI formulations is the upper limit dose thatthey can deliver, 500-600 μg/dose. Common pMDI doses are 100-300μg/dose⁵. Thus, a major goal for pMDIs has been to raise the dosage inorder to allow for pMDI delivery of less potent actives.

TABLE 5 ACI results for different protein pMDI formulations at differentprotein concentrations. Bovine serum albumin (BSA) and lysozyme (Lys)formulations shown. TED FPF (mg) (%) MMAD/GSD BSA (50 mg/mL)* 2.59 ±0.44 64 ± 3 2.48 ± 0.22/3.89 ± 0.11 Lys (20 mg/mL)*  3.97 ± 0.825 64 ± 92.49 ± 0.18/4.33 ± 0.59 Lys (50 mg/mL)** 3.81 62 2.62/3.56 Lys (70mg/mL)** 3.35 60 2.72/3.00 *HFA 227 **HFA 134a

To prepare the protein pMDI formulations, the protein powders are placedin 50 mL PYREX® beakers and pre-cooled in a −80° C. freezer. Thepropellant, preferably a hydrofluoroalkane propellant, is pre-cooled to−80° C. and then poured into the beaker containing the protein powdersto form suspensions ranging in concentrations from 0.7-7.4% w/w. Theresultant protein suspensions are placed in a dry ice/acetone bath andsonicated for two minute using a Branson Sonifier 450 (BransonUltrasonics Corporation, Danbury, Conn.) with a 102 converter and tipoperated in pulse mode at 35 W. 11 mL of the cooled protein formulationsare dispensed into 17 mL glass aerosol vials (SGD, Paris, France) andfitted with metering valves containing 100 μL metering chambers (DF10 RC150, Valois of America, Inc., Congers, N.Y.) using a compressor pump(Pamasol Model P2005, Pfaffikon, Switzerland). The vials are allowed towarm up to room temperature. Small amounts of lubricants (2-8% w/w),such as polysorbate 20 and polysorbate 80, may be added to theformulation prior to sonication to minimize clogging of the valve duringactuation due to the highly concentrated suspensions.

The highly concentrated protein pMDI's demonstrate desirable aerodynamicproperties ideal for pulmonary drug delivery. Impaction studies wereconducted with a non-viable eight-stage cascade impactor(Thermo-Andersen, Smyrna, Ga.) with an attached Aerochamber Plus® ValvedHolding Chamber (Trudell Medical International, London, Ontario, Canada)at a flow rate of 28.3 L/min to quantify total emitted dose (TED), fineparticle fraction (FPF), mass median aerodynamic diameter (MMAD), andgeometric standard deviation (GSD). FPF was defined as the percentage ofparticles with an aerodynamic diameter less than 4.7 μm. Threeactuations are expelled as waste prior to measurements. One actuation ismade into the ACI for analysis. The valve stem, actuator, and impactorcomponents are placed into separate containers with a known volume ofdeionized (DI) water. Each component soaks for at least 30 minutes toensure complete protein dissolution. The protein concentrations arequantitated with a Micro BCA Protein Assay manufactured by Pierce(Rockford, Ill). The absorbance of the solutions was measured at 562 nmusing the μQuant Model MQX200 spectrophotometer (Biotek InstrumentsInc., Winooski, Vt.). Untreated protein was used to prepare the proteinstandards at concentrations between 2 and 40 μg/mL. ACI results yieldedaverage TED's between 2.5-4.0 mg protein/actuation, average FPF'sbetween 56-64%, and average MMAD's between 2.5-2.8 (GSD's between3.0-3.9).

The ability of a pMDI to deliver a consistent dose is mandatory for thedelivery of sufficient and safe drug doses to patients. Thus, stablepMDI suspensions are desired to ensure dose uniformity. No visiblecreaming or settling was observed for the suspensions over 48 hours.FIG. 47 is an optical image of protein pMDI formulations (Lys in HFA 227with a drug loading of 20 mg/mL, Lys in HFA 134a with a drug loading of40 mg/mL, 50 mg/mL, 90 mg/mL, and BSA (BSA) in HFA 227 with a drugloading of 50 mg/mL, left to right) 4 hours after shaking.

Dose uniformity of the highly concentrated protein pMDI's isdemonstrated by actuating the pMDI through the firing adaptor of adosage unit sample tube (26.6×37.7×103.2 mm; 50 mL volume; JadeCorporation, Huntingdon, Pa.). A known volume of DI water is added todissolve the protein and the sampling tube is shaken and allowed to sitfor at least 30 minutes to ensure complete protein dissolution. Theprotein concentration is determined using the Micro BCA protein assay inconjunction with the μQuant spectrophotometer. The pMDI canister isweighed before and after each actuation to assure that the proper dosewas released.

TABLE 6 is a table of the dose uniformity results for different proteinpMDI formulations at different protein concentrations. % Theoretical isthe percentage of the theoretically loaded dose that is emitted duringactuation.

TABLE 6 DDV (mg/actuation)/% Theoretical BSA (50 mg/mL)* 3.65 ± 0.37/73 Lys (20 mg/mL)* 1.03 ± 0.058/~50 Lys (40 mg/mL)** 3.44 ± 0.286/~86 Lys(50 mg/mL)** 4.11 ± 0.110/~82 Lys (70 mg/mL)** 4.35 ± 0.274/~62 Lys (90mg/mL)** 2.94 ± 0.286/~33 *HFA 227 **HFA 134a

Further characterization of protein particles after aerosolization fromthe pMDI device is performed. The aerosolized particles are measured bylaser light scattering. Each formulation is actuated once through theACI spacer and throat. The aerosol exits the outlet of the throat about5 cm above the laser of the Malvern Mastersizer S (Malvern Instruments,Ltd., Worcestershire, UK). For each formulation 100 measurements of theaerosolized spray are made about every 5 ms. The recorded measurementsare averaged to give a final profile of the aerosolized particles on avolume basis. Scanning electron microscopy (SEM) images of aerosolizedparticles are also used to determine the size of aerosolized particles.Particles are collected from stage 3 of the ACI. Double carbon adhesivetape is applied to stage 3. The impaction test is conducted according tothe parameters mentioned earlier and the carbon tape is applied to analuminum SEM stage. The sample is sputter coated with gold-palladium for30 seconds using a K575 sputter coater (Emitech Products, Inc., Houston,Tex.). Micrographs are taken using a Hitachi S-4500 field emissionscanning electron microscope (Hitachi Ltd., Tokyo, Japan) at anaccelerating voltage of 5-10 kV. Particle images are sized on the SEMmicrographs using imaging software (Scion, Frederick, Md.). At least 50particles were measured for each formulation. Particle sizes from theSEM micrographs correlate well with sizes reported by laser lightscattering.

FIG. 48 is a SEM micrographs of aerosolized Lys particles (Lys in HFA134a pMDI loaded at 50 mg/mL). Aerosolized particles have geometricdiameters between 8-10 μm (FIG. 48A) and exhibit porous morphology (FIG.48B) and (FIG. 48C). Other formulations show similar morphologies.Aerosolized particle densities are also determined from the SEMmicrographs. The calibrated aerodynamic diameter of particles depositedon stage 3 of the ACI is 3.3-4.7 μm. Thus an average MMAD of 4.0 μm wasassumed for particles deposited on stage 3 of the ACI. Using therelationship, d_(a)=d_(g)(ρ_(g)/ρ_(a))^(0.5) (where d_(a) is theaerodynamic diameter, d_(g) is the geometric diameter, ρ_(g) is thedensity of the particle, and ρ_(a) is 1 g/cm³) and using the estimatedMMAD and geometric diameter (from the SEM micrographs), the density ofthe aerosolized particle is calculated. The low calculated densities(0.14-0.23 g/cm³) indicate that the aerosolized particles are highlyporous, which is expected because of the porous morphology observed inthe SEM micrographs. The low densities explain why the particles areable to reach deep lung levels despite a geometric diameter of 8-10 μm.

TABLE 7 is a table that illustrates the measured particle diameters foraerosolized protein particles. D_(v,50) (diameter at which thecumulative sample volume was under 50%) values were reported by Malvern.

TABLE 7 SEM Volume Average D_(v, 50) (μm) Diameter (μm) ρ (g/cm³) BSA(50 mg/mL)* 10.05 ± 0.01  10.75 ± 2.07  0.14 Lys (20 mg/mL)* 8.07 ± 0.068.67 ± 2.00 0.21 Lys (50 mg/mL)** 8.78 ± 1.68 0.21 Lys (70 mg/mL)** 8.41± 1.70 0.23 *HFA 227 **HFA 134a

Preparation of TFF ITZ particles. ITZ (about 500 mg, Hawkins, Inc.,Minneapolis, Minn.) was dissolved in about 40 mL of 1,4-dioxane (FisherChemicals, Fairlawn, N.J.). To the drug solution, 100 mL of t-butanol(Fisher Chemicals, Fairlawn, N.J.) was added. The ITZ in 1,4dioxane-t-butanol drug solution was passed at a flow rate of 4 mL/minthrough a 17 gauge (1.1 mm ID, 1.5 mm OD) stainless steel syringeneedle. The droplets fell from a height of 10 cm above a rotatingstainless steel drum (12 rpm) 17 cm long and 12 cm in diameter. Thehollow stainless steel drum was filled with dry ice to maintain a drumsurface temperature of about 223 K. On impact, the droplets deformedinto thin films and froze. The frozen thin films were removed from thedrum by a stainless steel blade and transferred to a 400 mL PYREX®beaker filled with liquid nitrogen. The excess liquid nitrogen wasevaporated in a −80° C. freezer. A Virtis Advantage Lyophilizer (TheVirtis Company, Inc., Gardiner, N.Y.) was used to dry the frozenslurries. Primary drying was carried out at −30° C. for 36 hours at 300mTorr and secondary drying at 25° C. for 24 hours at 100 mTorr. A 12hour linear ramp of the shelf temperature from −30° C. to +25° C. wasused at 100 mTorr.

Crystallization of TFF ITZ particles: FIGS. 49A-49C are optical imagesof the TFF ITZ particles (about 110 mg) were loaded as dry powder intoglass vials (SGD, Paris, France) and fitted with metering valves (DF10RC 150, Valois of America, Inc., Congers, N.Y.) using a Pamasol ModelP2005 compressor pump (Pamasol Willi Mader A G, Pfaffikon, Switzerland)(FIG. 49A). FIG. 49A is a photograph of 110 mg TFF ITZ powder loadedinto a glass vial. FIG. 49B is a photograph of a 10 mg/mL TFF ITZsuspension produced after addition of 11 mL of HFA 227 to FIG. 49A, andFIG. 49C is a photograph of 110 mg TFF ITZ powder after exposure to HFA227. 1,1,1,2,3,3,3-heptafluoropropane (HFA 227, Solvay, Greenwich,Conn.) was loaded into the vials containing drug using Pamasol fillingequipment (Model P2008) to yield a 10 mg/mL milky suspension (FIG. 49B).The pressurized suspensions may be referred to as pressurized metereddose inhalers (pMDIs). To collect TFF ITZ powder after HFA exposure, thepMDI was cooled in a −80° C. freezer, well below the HFA 227 boilingpoint of −16° C. Once the HFA was sufficiently cooled, the meteringvalve was removed and the HFA was allowed to warm in a dry box (relativehumidity<20%) until it completely evaporated (FIG. 49C). Exposure of TFFITZ powder to 2H,3H perfluoropentane (HPFP), a non-volatile surrogatefor HFA 227, was also studied.

Product Description and Characterization: Sub-micron amorphous particlesof a poorly water soluble drug, itraconazole (ITZ), were produced bythin film freezing (TFF), a particle formation process described inmanuscripts by Engstrom et al. and Overhoff et al.

FIG. 50 is a graph of the X-ray diffraction (XRD) pattern of ITZ beforeand after exposure to HFA 227. Crystallization of TFF ITZ particlesafter HFA exposure was determined using x-ray diffraction (XRD) anddifferential scanning calorimetry (DSC). XRD patterns and DSC scans ofTFF ITZ powder before contact with HFA were characteristic of amorphousmaterials (FIGS. 50-51). However, characteristic peaks of crystallineITZ were detected in the XRD profile after TFF ITZ particles wereexposed to HFA 227 (FIG. 50).

FIG. 51 is a graph of the Modulated differential scanning calorimetry(mDSC) of TFF ITZ powders before and after exposure to HFA 227 and HPFPand pure ITZ. DSC scans showed complete crystallization of the TFF ITZparticles after exposure to HFA 227, based on the absence of anendothermic recrystallization peak (FIG. 51). Similar results wereobtained after exposure of TFF ITZ particles to HPFP (FIGS. 50-51).These results are significant because crystallization of TFF ITZ may beinduced with a solvent that can be handled under atmospheric conditions.

To confirm the complete crystallization of TFF ITZ particles afterexposure to HFA, dissolution studies were conducted on the HFA-exposedTFF ITZ powder in pH 7.4 phosphate buffer (0.02% w/v SDS). Theequilibrium solubility of crystalline ITZ in the dissolution media wasexperimentally determined to be 1.4 μg/mL. HFA-exposed TFF ITZ powder (1mg) was added to 50 mL of dissolution media to yield an initial drugloading of 20 μg/mL. Sample aliquots (1.5 mL) were taken from thedissolution vessels at various time points. The aliquots were filteredimmediately using a 0.2 μm syringe filter. Dissolved drug levels did notsignificantly exceed equilibrium solubility of crystalline ITZ,suggesting that the HFA-exposed TFF ITZ particles were crystalline (FIG.52). FIG. 52 is a graph of the dissolution profile of TFF ITZ particlesafter exposure to HFA 227 conducted in pH 7.4 phosphate buffer (0.02%w/v SDS). TFF ITZ powder that had been previously exposed to HFA 227 (1mg) was added to the dissolution media (50 mL) to achieve an initialloading of 20 μg ITZ/mL. All samples were filtered with 0.2 μm pore sizefilters. The dashed line represents the solubility of “as received” ITZin the dissolution media.

FIG. 53 in a scanning electron microscopy (SEM) images of TFF ITZ (FIG.53A) before and (FIG. 53B) after exposure to HFA 227 and (FIG. 53C) SEMimage of TFF ITZ after pMDI was actuated into water, without anyexposure to air. Additionally, a change in morphology of the TFF ITZparticles before and after exposure to HFA 227 was detected by scanningelectron microscopy (FIG. 53A-53B). TFF ITZ particles prior to HFAcontact were spherical in shape. However, thin, plate-like structureswere observed after exposure to HFA. To further verify that thecrystallization of TFF ITZ was induced by HFA, a pMDI containing TFF ITZwas actuated into water, with the metering valve submerged below theliquid level, to produce a slightly turbid dispersion. The TFF ITZparticles emitted from the pMDI were collected by freezing andlyophilizing this dispersion. SEM images of the actuated TFF ITZparticles revealed thin, plate-like structures strongly resembling theparticles produced after HFA evaporation (FIG. 53C). Therefore, completecrystallization of amorphous TFF ITZ particles occurred upon exposure toHFA 227.

FIG. 54 is a graph of the dynamic light scattering (DLS) measurements ofHFA-exposed TFF ITZ in water. The sizes of 66% of the particles (byvolume) were 737 nm or less. Furthermore, dynamic light scattering (DLS)measurements show that TFF ITZ particle dimensions remained below 1 μmafter crystallization, with 66% of the particles by volume with ahydrodynamic radius of 737 nm or less (FIG. 54).

Production of TFF ITZ/BSA compositions. Two compositions using a 10/1and 5/1 ITZ/BSA ratios were formulated to demonstrate that a watersoluble component could be added to the poorly water soluble TFF ITZ toaid in wetting during dissolution. A 5 mg/mL loading was tested. Theresultant pMDI formulations were milky, white and uniform, and similarto the other TFF pMDIs.

Table 8 shows the results obtained from the Andersen Cascade Impactor.

Respriable Dose/Act (μg) % FPF MMAD (μm) TFF Itz 525 ± 23 56 ± 3 3.8 ±0.3 (10 mg/mL) Milled Itz 300 nm 29 ± 8 15 ± 3 6.0 ± 0.7 (10 mg/mL) TFFItz/BSA (10/1) 238 ± 5  67 ± 2 1.4 (5 mg/mL) TFF Itz/BSA (5/1) 302 701.5 (5 mg/mL)

FIG. 55A shows the scanning electron microscopic image of aerosolizedTFF ITZ and FIG. 55B shows the SEM image of the aerosolized TFF ITZ indissolution media at 37° C. after 1 minute. The study was conducted inphosphate buffer pH 7.4 containing 0.2% w/v SDS.

FIG. 56 is a plot showing the dissolution profiles of aerosolized TFFITZ and aerosolized milled ITZ particles (300 nm) studied in phosphatebuffer (pH=7.4) containing 0.2% w/v SDS at 37° C. The graph shows a muchmore rapid dissociation and dissolution of the aerosolized aggregateinto constituent particles in comparison to the milled ITZ particles.

The flocculated particles used for pMDI delivery may also be applicablefor dry powder inhalation. In a dry powder inhaler, shear forcesgenerated during inspiration break up the flocs to an appropriateaerodynamic size for deep lung delivery. The particles may be producedby either milling, controlled precipitation (CP), or TFF. Poorly watersoluble drugs, itraconazole (ITZ) and cyclosporine A (CsA), and watersoluble proteins, bovine serum albumin (BSA) and lysozyme (lys), werethe model drugs used to demonstrate DPI delivery of nanoparticlesproduced by CP and TFF. Drug powders were aerosolized and characterizedusing either an Aerosizer/Aerodisperser (TSI, Shoreview, Minn.) or anAPS 3321/3343 (TSI, Shoreview, Minn.) disperser.

FIG. 57 is a graph of the aerodynamic diameters of milled, TFF, and CPdrug compositions measured by the APS 3321/3343 and theAerosizer/Aerodisperser systems. VMAD is the volume mean aerodynamicdiameter. The drug compounds studied include the poorly water solubledrugs itraconazole (ITZ) and cyclosporine A (CsA), as well as bovineserum albumin (BSA) and lysozyme (lys). T80 and T20 are the surfactantstween 80 and tween 20 (Sigma Chemical, St. Louis, Mo.).

The aerosolized CP and TFF powders possessed aerodynamic diameterspredominantly between 2.0-3.5 μm, on a volume basis, ideal for pulmonarydelivery, as seen in FIG. 57. These diameters are in a range that isknown to be desirable for efficient deep lung delivery. Furthermore, thesizes are in good agreement for the two dispersers. Only twocompositions containing low melting point stabilizers, such as Tweensurfactants, possessed aerodynamic diameters larger than 8 μm.Compositions containing both a poorly water soluble drug (Itz) and aprotein (BSA) were also shown to yield optimal aerosol particles forpulmonary delivery.

FIG. 58 is a graph of the aerodynamic particle size distribution for theTFF lys composition. VMAD is the volume averaged mean aerodynamicdiameter and GSD is the geometric standard deviation.

An example of the aerodynamic particle size distribution for theaerosolized particles is shown in FIG. 58 for the aerosolized TFF lysformulation. SEM micrographs of TFF lysozyme powder beforeaerosolization and after aerosolization are shown in FIG. 59A-C.Lysozyme particles produced by TFF have a morphology of small nanorods,with lengths ˜500 nm and diameters between ˜50-100 nm, as seen in FIG.59A-C. Aerosolization of the powder disperses the nanorod floc to yieldaerosol particles roughly 3 micron in diameter. High magnificationimages of the aerosolized particles show that the rod-shaped primaryparticles are maintained throughout the aerosolization process. The SEMmicrographs of the aerosolized TFF lys particles were obtained byplacing ˜25mg of powder in gallon sized Ziploc bag. Double sided carbontape was placed onto the inside of the bag. The opening of the bag wasthen rubber banded around the nozzle of a can of compressed air. A shortburst of air was actuated into the bag to disperse the powder. Thecarbon tape was removed from the inside of the bag and placed onto anSEM stage for microscopy.

The present invention provides a novel composition and method of makingcompositions for the development of a dry powder inhaler (DPI) systemcomprised of highly dispersible and deformable nano-structuredaggregates that may be templated by air to form aerosol particlesappropriate for pulmonary delivery. The nano-structured aggregatesconsist of a porous web of drug nanoparticles, in which the primaryparticles making up the web are touching one another but are not tightlypacked. The sizes of the individual webs may range from several micronsto several hundred microns, as observed by scanning electron microscopy.The primary drug particles may be spherical (aspect ratio near or equalto 1) or elongated (aspect ratio greater than 1) in shape. Thenanoparticle web is considered deformable because, upon entrainment inair, portions of the nanoparticle web may be sheared off by design intosmaller aggregates. These smaller aggregates possess aerodynamicdiameters appropriate for deep lung delivery (2-5 μm), as determined bytime-of-flight measurements. Thus, the final aerosolized particle is“templated by air,” as the air stream provided by a patient'sinspiration through a DPI device is capable of providing the forcenecessary to shear off a portion of the nanoparticle web to form aninhalable particle. Highly dispersible and deformable nano-structuredaggregates of itraconazole (ITZ), bovine serum albumin (BSA), andITZ/BSA nanoparticles have been produced. This approach is applicable toother proteins, gene delivery, peptides and low molecular weight drugsof a range of water solubilities.

Until recently, the delivery of protein therapeutics has been largelylimited to parenteral delivery due to the chemical and physicalinstabilities of proteins and challenges in permeating biologicalmembranes (79) Among the non-invasive routes, pulmonary delivery offersadvantages of large alveolar surface area (˜100 m²), rapid absorptionacross the thin alveolar epithelium (0.1-0.5 μm), avoidance of firstpass metabolism, and sufficient bioavailabilities (79-87). DPIs are thenewest form of pulmonary mode of administration of active pharmaceuticalingredients and have become a popular delivery method for drugs,especially proteins, because delivery and storage as a dry powder isdesirable in terms of stability. Because optimal aerodynamic propertiesare crucial to deep lung deposition, particle aggregation andinefficient powder dispersion, common problems observed in DPIs, aredetrimental to DPI performance. Attempts to optimize particleinteractions within a DPI formulation have included the addition ofcarrier particles to improve powder flow properties, and themodification of particle shape, size, surface roughness, or surfaceenergy by the addition of low surface energy particles (88).

The nano-structured aggregates readily deform and disperse intooptimally sized particles for pulmonary delivery upon entrainment withair without the need for carrier particles and minimal amounts, and insome cases, no surfactant, which facilitates the production of highpotency drug powders. Traditional DPI powders consist of pre-formed 1-10μm particles. To achieve high fine particle fractions (FPF), the DPIdevice must efficiently deaggregate the pre-formed powder particles downto primary particles. Particle properties such as shape and surfaceroughness strongly influence dispersion characteristics, andconsequently FPF and aerodynamic properties, of drug powders from a DPI(88). Furthermore, traditional DPI particles typically experience strongattractive Van der Waals (VDW) forces when packed into a DPI device dueto short separation distances between particles (estimated to be on theorder of 5 nm (89)), which impedes efficient particle deaggregation.Unlike conventional DPIs, this invention does not require the DPI deviceto deliver pre-formed powder particles comprising drug, but moreadvantageously aggregates of the primary drug particles. By loading aporous web of nanoparticles into a DPI device, common DPI problemsobserved with pre-formed particles, such as mechanical interlocking andhigh surface energies between particles, are minimized due to the porousstructure of the nanoparticle aggregates, which experience a reducedattractive VDW force compared to dense micron-sized particles (89-91).Thus, the nano-structured aggregates are easily sheared and deformedinto respirable aggregates with aerodynamic diameters (d_(a)) between2-5 μm due to the extremely weak Van der Waals forces holding the porousaggregate together.

This invention provides an efficient way to deliver nanoparticles to thelungs using a DPI. Optimal aerodynamic behavior has been achieved bydelivering highly dispersible and deformable, porous aggregates ofnanoparticles as a dry powder for enhanced pulmonary delivery. Pulmonarydelivery to replace other methods such as parenteral and oral delivery.

There are possible challenges concerning the stability of the drugpowder in a compacted state (when loaded into a DPI device) over longperiods of time. Long term storage may cause aggregation which mayaffect the ability of the powder to efficiently disperse into aerosolparticles optimal for pulmonary delivery. Minimal aggregation betweenparticles is anticipated for the porous nanoparticle aggregates, due toweak attractive VDW forces, which results from their porous structure.Strong attractive VDW forces between particles increase the possibilityfor irreversible aggregation, a problem for conventional DPIs (88). Inthe case that the drug particles aggregate and do not disperseefficiently after storage, components such as leucine may be added tothe formulation, which has been shown to enhance dispersability of drypowders for inhalation(88,92).

Dry powder inhaler (DPI) formulations typically consist of micronizeddrug blended with carrier particles packed into a bed (e.g., capsule,blister pack, etc). In order to achieve effective drug deposition in thelungs, the patient must generate sufficient shear and turbulence tofluidize the packed powder and carry the drug particles to the lungsupon inspiration. Thus, the ability of the packed drug particles todisperse into primary particles is essential for the aerosolized powderto possess optimal aerodynamic properties for deep lung deposition. Toenhance particle dispersion in DPIs, Langer and Edwards have producedlarge, porous particles (AIR® particles), with diameters between 5-20 μmand low tap densities (<0.4 g/cm³), which experience reduced Van derWaals attractive interactions compared to non-porous particles due totheir porous morhpology. Despite the large size of AIR® particles, theirlow porosity allows them to possess aerodynamic properties similar tothat of smaller, non porous particles.

In this invention, nano-structured aggregates have been shown to readilydisperse into optimally sized particles for pulmonary delivery uponentrainment with air without the need for carrier particles and minimalamounts, and in some cases, no surfactant, which facilitates theproduction of high potency drug powders. Unlike traditional micronizedDPI powders and AIR® particles, this invention does not require the DPIdevice to deliver primary drug particles, but aggregates of the primarydrug particles. The porous web of nanoparticles is easily sheared intoaggregates with aerodynamic diameters (d_(a)) between 2-5 μm due to theextremely weak Van der Waals forces holding the porous aggregatetogether.

The present invention provides a method of making brittle-matrixparticles through blister pack freezing using ultra-rapid freezing (URF)technology adapted for manufacture in a pharmaceutical blister pack. Forexample, the present invention can use ultra-rapid freezing using theblister packs contents and an ADVAIR DISKUS®. The device was opened, theblister strip was removed and peeled open. The contents (drug andlactose) of each blister pack were removed. The aluminum strip wascleaned with deionized water, rinsed with ethanol, and allowed to dryand room temperature.

The brittle-matrix particles were formed. Tacrolimus and lactose(TACLAC) and tacrolimus (TAC) solutions were prepared separately in 1 mLof ACN:water (3.2) each. Both solutions contained 0.75% w/v solids. APYREX® Petri dish was filled with liquid nitrogen, and blister packswere added. One at a time, blister packs were removed from the liquidnitrogen bath, and 25 μL of drug solution was added to the concaveindentation. The product was frozen immediately upon contact and placedin a −80° C. freezer. All frozen blisters were lyophilized according tothe recipe described herein. After lyophilization, all blister packswere stored under vacuum in a sealed desiccator.

Aerosol testing was conducted using a Next Generation PharmaceuticalImpactor (NGI) with coated collection surfaces. Carefully, a singleblister containing either TACLAC or TAC was added to the inhalationposition of the ADVIAR DISKUS®, and the device was resealed and mountedon the induction port of the NGI by a silicone molded fitting. Flow ratenecessary to achieve a 4 kPa pressure drop across the Diskus wasdetermined to be 66 L/min; therefore, all studies were conducted at thisflow rate. Both TACLAC and TAC blister formulations were actuated threetimes before collection of the impacted formulation from the stages.Rinsing and high performance liquid chromatography (HPLC) method forquantification of drug was performed. Fine particle fraction is definedas the percentage of drug mass emitted that is below 5 μm in diameter.

FIG. 60 is an aerodynamic distribution of brittle-matrix particlesemitted from an ADVAIR DISKUS®. FIG. 60 shows the aerodynamicdistribution of both formulations analyzed and the FPF measured forTACLAC and TAC formulations were 35.1% and 19.8%, respectively. Clearly,the shear imparted by the Diskus device is not sufficient to obtain thequantity of highly respirable particles made by the HANDIHALER®.Although FPFs measured in this initial study were low in comparison,brittle-matrix particles tested here still outperformed the formulationsmarketed with the Diskus. The FLUTIDE DISKUS® was evaluated forefficiency in a study by Steckel in 1997, where only 25.4% of theemitted dose was in the aerodynamic range below 6.4 μm (92). TACLAC,when prepared by blister freezing, resulted in an aerosol with 41.0% ofthe emitted dose below 6.4 μm.

The total emitted dose (TED) for TACLAC and TAC formulations were 78.6%and 97.3%, respectively. It was apparent during formulation productionthat temperature of the blisters packs determined the shape andmorphology of the frozen formulation because of the effect on the rateof freezing. This may have contributed to some of the difference in TEDbetween formulations, as TACLAC blisters were thought to be warmer uponaddition of the drug solution. It was also observed that even uponstorage in a vacuum desiccator, the hydroscopic effects of lactosecaused “collapse” of TACLAC particles. Cohesion caused by moisturesorption could also have caused increased retention of TACLAC in theblister.

As used herein, the term “surfactant” means a substance that reduces thesurface tension of a liquid, thereby causing it to spread more readilyon a solid surface. Examples of surfactants for use with the presentinvention, include, all surfactants suitable for administration to thelungs, including sodium salts of cholate, deoxycholate, glycocholte andother bile salts; Span 85, Lauryl-beta-D-maltoside, palmitic acid,glycerol trioleate, linoleic acid, DPPC oleyl alcohol, oleic acid,sodium oleate, and ethyl oleate.

Non-limiting examples of the active agents of the present inventionincludes antifungal agents having one or more of azoles and/orallylamines, e.g., natamycin, flucytosine, miconazole, fluconazole,itraconazole, clotrimazole, econazole, miconazole, ravuconazole,oxiconazole, sulconazole, terconazole, tioconazole, fenticonazole,bifonazole, oxiconazole, ketoconazole, isoconazole, tolnaftate,amorolfine, terbinafine, voriconazol, posaconazol, or thepharmacologically acceptable organic and inorganic salts or metalcomplexes or mixture thereof.

Delivery of the present invention to the lung can be achieved throughany suitable delivery means, including a nebulizer, a dry powderinhaler, a metered dose inhaler or a pressurized metered dose inhaler.The suitable delivery means will depend upon the active agent to bedelivered to the lung, the desired effective amount for that activeagent, and characteristics specific to a given patient.

In addition, the present invention may include one or more excipientsthat modify the intended function of the effective ingredient byimproving flow, or bio-availability, or to control or delay the releaseof the effective ingredient, e.g., nonlimiting examples include: Span80, Tween 80, Brij 35, Brij 98, Pluronic, sucroester 7, sucroester 11,sucroester 15, sodium lauryl sulfate, oleic acid, laureth-9, laureth-8,lauric acid, vitamin E TPGS, Gelucire 50/13, Gelucire 53/10, Labrafil,dipalmitoyl phosphadityl choline, glycolic acid and salts, deoxycholicacid and salts, sodium fusidate, cyclodextrins, polyethylene glycols,labrasol, polyvinyl alcohols, polyvinyl pyrrolidones and tyloxapol,cellulose derivatives, and polyethoxylated castor oil derivatives.

Other suitable solvents include but are not limited to: ethanol,methanol, tetrahydrofuran, acetonitrile, acetone, tert-butyl alcohol,dimethyl sulfoxide, N,N-dimethyl formamide, diethyl ether, methylenechloride, ethyl acetate, isopropyl acetate, butyl acetate, propylacetate, toluene, hexanes, heptane, pentane, 1,3-dioxolane, isopropanol,n-propanol, propionaldehyde and combinations thereof.

The preparation of particles and respirable aggregates using a URFmethod includes a solution of ITZ (0.0798 g) with pluronic F-127 (0.0239g) is prepared by loading the dry solids into a vial. A prepared 95/5 wt% blend of t-butanol and toluene (10.03 g) is loaded into the vial. Theresulting slurry is heated until a solution was formed. (68 to 70.degree. C.). The resulting solution is applied to the freezing surfaceof the URF unit, which had been cooled to −78.degree. C. over athree-minute time period. The frozen solvent, drug, and excipient matrixis collected in a tray, which had been cooled with dry ice, andtransferred into a 60-mL jar, which had been cooled with dry ice. Thejar containing the URF processed frozen solid is then placed on a freezedrying unit and lyophilized for approximately 17 hr at 100 mtorr. Afterlyophilization, 0.0700 g of the URF processed solid is recovered as adry flowable powder. The mean volume average particle sizes (with andwithout sonication) of the reconstituted drug particles are measuredusing a Coulter LS 230. The particles are amorphous.

Pulmonary inhalation of low-density porous particles enables deep lungdelivery with a more efficient dose and less dependence on device designand patient inspiration. The large geometric diameter of porousparticles enhances sustained in vivo drug release by avoidance ofphysiological clearance mechanisms. The present invention providesrespirable low-density microparticles (25-50 μm) produced in situ frombrittle drug matrices to achieve highly efficient deep lung delivery viaa dry powder inhaler. The brittle matrices comprising a solid dispersionof drug and excipient are sheared apart by a standard inhalation deviceto produce ultra low-density particles with appropriate aerodynamicdiameters (1-5 μm). Skeletal particle density for each formulationdetermined from the measurement of the geometric and aerodynamicdiameters were as low as 0.01 g/mL. In contrast, reported skeletaldensities of large porous particles produced by other techniquesare >0.05 g/ml and often >0.1 g/ml After incorporation of biocompatiblematerials such as pharmaceutical sugars into the formulations,aerosolization of the resulting brittle matrices produced fine particlefractions (FPF) as high as 70.3% and total emitted doses (TED)consistently higher than 95%. Accuracy of aerodynamic testing withcascade impaction was improved markedly by coating the collectionsurfaces. The aerosolization of the particles was found to besusceptible to humidity induced capillary forces and electrostaticcharging, although formulations containing mannitol or no sugarexcipient proved to be more robust. Under completely dry conditions, theformulation made with anhydrous lactose exhibited improved brittlefracture and aerosolization, showing a 10% increase in FPF and 0.8 μmdecrease in mass median aerodynamic diameter (MMAD) relative to the sameformulation stored at 50% RH. Low-density microparticles, produced fromaerosolization of brittle matrices produced by thin film freezing (TFF)exhibit exceptional respirable properties and may prove to be a usefulplatform for highly efficient delivery of thermally labile, highlypotent, and poorly soluble drugs.

TFF technology was employed for the production of dry powders. Briefly,a cosolvent mixture of acetonitrile (ACN) and water was used to dissolvetacrolimus and sugar excipient. Tacrolimus and lactose (TACLAC),tacrolimus and mannitol (TACMAN), tacrolimus and raffinose (TACRAF), andtacrolimus without a sugar excipient (TAC) were dissolved in thecosolvent solution. The ratio of tacrolimus to excipient was 1 to 1 andeach solution prepared for TFF had a total solids concentration of 0.75%w/v. The solutions were rapidly frozen on a cryogenically cooled (<−50°C.) stainless steel surface and then maintained in the frozen state inliquid nitrogen. A detailed description of the TFF process is given byOverhoff et al and Engstrom et al. Solvents were sublimated bylyophilization using a VirTis Advantage Tray Lyophilizer (VirTis CompanyInc., Gardiner, N.Y.), leaving a drug and sugar solid dispersion in drylow-density particles. Lyophilization was performed over 40 hours atpressures less than 200 mTorr while the shelf temperature was graduallyramped form −60° C. to 25° C. Product was removed form the lyophilizerafter dry N₂ was bled into the chamber to equilibrate to atmosphericpressure. Product was quickly covered in order to prevent ambienthumidity from affecting the formulation. Powders were stored in atransparent vacuum desiccator at room temperature.

Bulk and tapped density of TFF produced powders were measured accordingto a method adapted from USP method I using a Varian Tapped DensityTester (Varian, Palo Alto, Calif.). An adaptation was made due to thelimited supply of powder for testing where a 100 mL graduated cylinderwas replaced by a 5 mL graduated cylinder. Hausner ratio and Can's(Compressibility) index were calculated for each formulation based onUSP guidelines. Additionally, skeletal densities of dispersed powderswere calculated based on measured aerodynamic and geometric diameter forcomparison to measured density values. Calculations were performed,where the dynamic shape factor (X) was assumed to be 1.5 for alldispersed powders. Mass median aerodynamic diameter (MMAD) wasdetermined based on all particles emitted from the device for thesecalculations.

Geometric diameter of TFF produced aerosolized and non-aerosolizedpowder was determined by low angle light scattering with an inhalationcell and an induction port. A HANDIHALER® (Boeringher Ingelheim GmbH,Ingelheim am Rhein, Germany) containing a size 3 hypromellose (HPMC)capsule was secured to the mouth of the induction port by a moldedsilicone adapter. Aerosolization of powder was achieved at a flow rate51 L/min, providing a 4 kPa pressure drop across the device. Dataacquisition took place over 4 seconds and only when laser transmissiondropped below 95%. Non-aerosolized powder diameter was measured byadding powders to the opening of the inhalation cell without theinduction port and without air flow.

A Next Generation Pharmaceutical Impactor (NGI) (MSP Corp., Shoreview,Minn.) was used to determine aerodynamic properties of low-densitymicroparticles. A HANDIHALER® containing size 3 capsules andapproximately 3 mg of formulation was attached to the induction port bya molded silicone adapter. All tests, with the exception of thoseinvestigating the influence of gelatin capsules on aerodynamic diameter,were conducted with size 3 HPMC capsules. Aerosols were produced over 4seconds at a flow rate of 51 L/min. Stage cut size diameters werecalculated to be 8.8, 4.9, 3.1, 1.8, 1.0, 0.6, 0.4, and 0.2 μm forstages 1 through 7 and micro-orifice collector (MOC), respectively (24).In most impaction tests run, collection surfaces were coated with 1%Tween 80 in ethanol, which is one of many coating materials recommendedby the European Pharmaceutical Aerosol Group (EPAG). Tween solution wasapplied to each collection surface (approx 1 mL) and allowed to dry for1 hour. After aerosolization, collection of deposited powders wasaccomplished by rinsing with 2, 5, 10, and 2.5 mL mobile phase for thedevice, induction port, pre-separator (if used), and stages 1-MOC,respectively. The pre-separator is designed to collect coarse particles(>15 μm) before the enter the body of the NGI and was included only whencoarse lactose is used. High performance liquid chromatography (HPLC)and a method for tacrolimus detection were used to quantify thecollected drug from each rinsing.

Total emitted dose (TED) of each test was calculated as the percentageof dose emitted over total dose assayed. Fine particle fraction (FPF)and MMAD were calculated using Sigmaplot 2000 (Systat Software Inc, SanJose, Calif.) to fit a 3 parameter logistic curve to plotted data. MMADand geometric standard deviation (GSD) were calculated based on drugdeposition on stage 1 through MOC, while FPF was calculated based on TEDand represent the percentage of particles with an aerodynamic diameterless than 5 μm.

Water sorption profiles were determined for brittle matrix powdersmanufactured by TFF using Dynamic Vapor Sorption (DVS-1). For eachformulation, glass sample cells were filled to capacity (0.5 mL)resulting in weights ranging from 5 to 30 mg, depending of particledensity. Samples were dried with nitrogen gas until a baseline wasestablished with less than 0.002% change in dm/dt. Each formulation wasrun for a complete sorption/desorption cycle between 0 and 90% relativehumidity (RH). Humidity was increased/decreased by 5% after equilibriumwas reached, as determined by a dm/dt less than 0.002%. Sorptionisotherms were calculated and plotted according to percent change inmass minus the initial dry formulation weight. DVS was also used tocreate a controlled humidity environment for powder dispersion to betested using laser scatter. Humidities of 90, 50, 20, and 0% wereexposed to powder formulations for 30 minutes in succession. Equilibriumwas assumed after 30 minutes, and an aliquot of powder was removed fortesting. All testing began with 90% humidity so that skeletal densitychanges due to hygroscopicity would be applied to subsequent samplestaken at 50, 20, and 0% RH.

TFF technology produces low-density pharmaceutical matrices, oftencontaining amorphous drug, stabilized with high T_(g) excipients. Inprevious reports, TFF has been used as a particle engineering technologyto enhance the aqueous solubility of poorly water soluble drugs for oraland pulmonary applications. Through stabilization of amorphous drugmorphologies with glassy excipients, inclusion of hydrophilic materials,and increased surface area, TFF manufactured powders have been shown tooffer improvements in wetting, dissolution rates, and solubility,leading ultimately to increased bioavailability. Given the desirableattributes of these powders and the efficiency of low-density powdersfor deep lung delivery, we hypothesized that these particles wouldresult in superior aerosol performance relative to previously researchedporous particles made by traditional manufacturing techniques. In oneformulation, drug and excipient are present in a one-to-one ratio in asolid dispersion. SEM samples analyzed by EDX reveal a homogeneousdispersion of tacrolimus and lactose, indicated by the presence ofnitrogen. Other studies have produced amorphous powders with TFF andhave shown through x-ray diffraction (XRD) patterns and differentialscanning calorimetry (DSC) that these dispersions often form solidsolutions.

For effective delivery of respirable low-density microparticles, apassive inhalation device with the ability to produce high shearvelocities is required. Fortunately, most device designs already requireturbulent, high shear airflow to provide adequate force for theseparation of micronized drug from carrier lactose. The HANDIHALER®, asingle dose capsule-based DPI, was chosen for aerosolization of brittlematrices in this study. Through a patient induced pressure drop,contents of a size 3 capsule within the device are released by flowwithin and around the capsule. Prior to discussion of furtherformulation considerations, aerosol performance dependence on capsulecomposition is first investigated. HPMC capsules produced a significantimprovement (P<0.05) in FPF over that of gelatin capsules while MMAD wasunchanged. The shape and area of the puncture hole created couldinfluence the velocity/turbidity of air entering and leaving thecapsule. In previous reports of puncture shape of gelatin and HPMCcapsules, it was concluded that more irregularly shaped holes wereformed in the less brittle HPMC capsules, relative to gelatin. Fordelivery of this formulation, a smaller, non-spherical puncture mayprovide a greater shear force than a large spherical opening, impartingfor fracture of friable matrices. Other non-aerodynamic advantages ofpowders released from HPMC capsules include low moisture content andincreased stability at elevated humidity.

Determination of friability of brittle matrix formulations was performedby comparing geometric particle distribution of low-density particlesemitted from the DPI device with that of “bulk” or non-aerosolizedmatrices. The effect of shearing induced by the HANDIHALER® wassubstantial as indicated by the difference between the volume momentmean (d_(4,3)) of bulk (502.4 μm) and DPI emitted (62.0 μm) particles.The volume moment mean is a numerical representation of the “center ofgravity” of a volumetric distribution, also known as the De Brouckeremean diameter. Because particle fracture is vital to the aerodynamicperformance of these particles, excipient selection focusing on materialproperties such as strength, brittleness, and hygroscopicity iscritical. The ability to fracture the bulk particles with air flow isconsistent with the fracture of large open friable flocs of similarparticles produced by TFF, in which shear was produced by ahydrofluoralkane in a pMDI. In each case, the shear produces particleswith proper aerodynamic and geometric diameters to achieve high fineparticle fractions. A major difference for the pMDI approach is that theparticles collapse as the HFA droplets evaporate. An additional caveatto formulation of dry powder for inhalation is that these excipients benontoxic and non-irritating for delivery to the lungs or otherwisegenerally recognized as safe (GRAS) by the FDA.

The influence of pharmaceutical sugars on aerosol performance wasdetermined by measurement of both geometric and aerodynamic properties.In this pulmonary delivery platform, the fundamental principle forproducing highly respirable microparticles relies on the brittlefracture of ultra low-density matrices to create small diameterparticles of the same structure and density. Accordingly, pharmaceuticalmaterials shown to experience brittle fracture under applied stress werechosen, such as those used in direct compression (DC) tabletting.Sacchirides used for DC are more likely to experience brittle fracturethan ductile cellulose excipients, and are more appropriate for ourapplication. In addition, some sacchirides are established as beingnon-irritating in the lungs. Two sacchirides, α-lactose and raffinose,were selected based on their brittle properties; however, the ability toinduce brittle fracture with a passive DPI device had not beendetermined. Mannitol, a less hygroscopic sugar alcohol, was alsoselected for evaluation as an excipient in brittle matrix powders. Afterproduction, initial visual observations of unpackaged product showedthat the skeletal structure of TACMAN and TAC were less susceptible toambient humidity than other formulations.

Aerodynamic evaluation of emitted low-density microparticles on a stagecoated NGI showed elevated TED and FPF when compared to traditional drypowder inhalation formulations (2). Initial testing of newly preparedformulation revealed TACLAC and TACRAF as the most efficientlyperforming aerosols, with FPF of 70.3 and 63.5%, respectively (Table 3).Distribution of deposition on within the NGI, shown in FIG. 5 a revealsa lower stage deposition of TACLAC and TACRAF in comparison with theother formulations. Assuming that all formulations have similar density,it could be concluded that increased particle fracture of particlescontaining anhydrous a-lactose and anhydrous raffinose resulted inimproved aerodynamic properties. Although some drawbacks to anhydrousmaterial exists (as will be discussed), complete water removal fromsacchirides often results in an significant increase in friability andbrittleness (38, 39). Specifically, anhydrous raffinose is noted for itsfriability and has been determined to be the “most fragile”pharmaceutical sugar (38). It is interesting to note that the morebrittle, anhydrous form of raffinose is also amorphous, contrasting withthe general conception that amorphous sugars are more ductile. Differingfrom raffinose, anhydrous α-lactose is similar to others excipients inthat the amorphous form is commonly less brittle than the crystalline.High TED for all formulated powders is indicative of the reduced surfacecohesion of low-density powders, normally caused by van der Waals,capillary, and electrostatic forces in traditional formulations;although, further analysis shows that these forces do still play a rolein particle dispersion.

Humidity had an inhibitory effect on the performance of TACLAC, mostlikely due to increased plasticity of the brittle matrix. TACMAN provedto benefit from additional moisture, as shown by an increase in FPF,perhaps due to reduction in electrostatic charging. FIG. 6 b shows thebimodal distribution indicative of electrostatic adhesion of TACMAN atlow RH. It can also be seen that TED decreased slightly in every 50% RHformulation, which could be expected due added formulation adhesion tothe capsule wall in the presence of moisture. Water sorption to powdersurfaces can both improve and hinder aerosol dispensability. Previousreports have shown that dry powder formulations stored at approximately60% RH maximize the drug FPF (40). In general, humidities >60% result incapillary forces predominating, while electrostatic charge remains low.Relative humidities <60% will cause elevated electrostatic adhesion ofpowders due to the lack of moisture-induced charge dissipation. Forbrittle matrices, presuming they are amorphous, the plasticizing effectof water must also be considered. Amorphous materials are particularlysusceptible to water plasticization, as is the case for anhydrouslactose and raffinose, which will result in reduced brittle fracture andcould lead to increased particle density due collapse of the matrixstructure.

Bulk and tap density testing, as defined by the USP, were used tocharacterize density of each powder formulation. While all densitiesmeasured were extremely low, the bulk density of

TACLAC was approximately twice that of the other formulations, mostlikely due to matrix water absorption and subsequent particlecontraction. Tap density was also measured and used to calculate Carr'sindex. Can's index, or compressibility index, is used to describe aductile material that undergoes plastic deformation or a brittlematerial that fractures under an applied force. Assuming that allchanges in powder density were due to brittle fracture, this dataprovided another indication that TACRAF is the most brittle of thepowders investigated, showing a Can's index of 50.

For comparison with USP density testing, correlation between sizedistribution data produced by cascade impaction (NGI) and laserdiffraction (SPRAYTEC®) analysis were also used to determinemicroparticle density. Knowing both the MMAD and the volumetric mediandiameter (D_([50])), equation 1 was used to calculate the skeletaldensity of the sheared microparticles exiting the DPI. Approximation ofthe shape factor was necessary due to its effect on aerodynamicdiameter, and was assumed to be 1.5. SEM images portrayed a jagged andirregular morphology of the aerosolized particles, similar that of asand particle, which has a dynamic shape factor of 1.57. Calculation ofparticle density proved to be slightly lower that measured by bulkdensity testing; however, comparing formulations to one another showed asimilar relationship. It is possible that a lower prediction based onemitted aerosols was due to non-emitted particles remaining in thedevice that were excluded from characterization. Relative to each other,TACMAN and TAC produced the lowest density particles, most likely due totheir non-hygroscopic nature, while TACRAF, and particularly TACLAC,showed higher density. Changes in skeletal densities of lactose, andperhaps raffinose, upon exposure to ambient moisture are due to theirtendency to adsorb water and could be explained by two mechanisms. It islikely that adsorbed water effectively plasticizes the fragile matrixcausing lowering of the glass transition temperature (T_(g)) andrelaxation of supporting structures, subsequent collapsing the particle.Increased mobility of amorphous material will also lead to formation ofa more thermodynamically stable, crystalline form. Powder collapse dueto low material T_(g) has been observed previously in sucroseformulations, where inclusion of Dextran-40 significantly increased theT_(g) and resulted in improved structural integrity and longerstability. By increasing T_(g) of the matrix material, molecularmobility would be limited resulting in reduction of particle shrinkageand crystal formation. Another possibility exists for particle collapseat high humidity (<65%) where the material becomes deliquescent,partially dissolving in adsorbed moisture and effecting the integrity ofthe particle. It is doubtful, however, that deliquescent dissolving oflactose is a viable cause for particle collapse since the criticalrelative humidity needed for this to occur is 99% RH. Mannitol andtacrolimus, being non-hygroscopic and hydrophobic, respectively, do notexperience noticeable changes in skeletal density over time due to lesswater adsorption and a higher T_(g).

Inhalation of low-density microparticles formed from brittle matriceswith a marketed DPI device is a viable platform for highly efficientdeep lung delivery of drugs. Unlike delivery strategies that utilizepreformed particles, the brittle matrix TFF powders are sheared intoextremely low-density (0.05-0.01 g/cm³) microparticles in situ bypatient inspiration. After incorporation of biocompatible materials suchas pharmaceutical sugars into the formulations, aerosolization of theresulting brittle matrices produced fine particle fractions (FPF) ashigh as 70.3% and total emitted doses (TED) consistently higher than95%.

Additional benefits of this platform for inhalation therapeutics includesolubility enhancement for amorphous particles, rapid dissolution forhigh surface area sub-500 nm primary structures, and the ability toformulate process-sensitive actives with TFF. Future studies focusing ondose consistency, in vivo characterization, and process scale up will beinvestigated to determine the viability of this platform as analternative to large porous particles and traditional carrier-basedformulation

The preparation of particles and respirable aggregates using acontrolled precipitation (CP) method includes a batch controlledprecipitation process. An aliquot of 1.77 grams of Brij 98 is dissolvedin 148.33 grams of deionized water. The aqueous solution is thenrecirculated, using a centrifugal pump (Cole-Parmer Model 75225-10) atmaximum pump speed (9000 rpm), through recirculation loop and throughheat exchanger (Exergy Inc. Model 00283-01, series heat exchanger) untilthe aqueous temperature is 5.degree. C. An aliquot of 30.19 grams of asolution containing 5 wt % ITZ in 1,3-dioxolane is added into therecirculating aqueous solution over about seconds, which results in thecontrolled precipitation of a particle slurry. The particle size of theparticle slurry is measured, without filtration or sonication, using aCoulter LS 230. The particle slurry is then fed to a wiped-filmevaporator having a jacket temperature of 40° C., an absolute pressureof 8 mm Hg, and a feed rate of 15 mL/min. The particle size of thesolvent-stripped slurry is measured, without filtration or sonication,using a Coulter LS 230.

Examples of active agents include, but are not limited to antibiotics;analgesics; anticonvulsants; antipyretics; anti-inflammatories;antitussive expectorants; sedatives; antidiabetics, antifungals,antiepileptics, antineoplastics; antiulcer agents; antiparkinsonianagents, antirheumatics, appetite suppressants, biological responsemodifiers, cardiovascular, agents, central nervous system stimulants,contraceptive agents, diagnostic agents, dopamine receptor agonists,erectile dysfunction agents, fertility agents, gastrointestinal agents,hormones, immunomodulators; antihypercalcexnia agents, mast cellstabilizers, muscle relaxants, nutritional agents, ophthalmic agents,osteoporosis agents, psychotherapeutic agents, parasympathomimeticagents, parasympatholytic agents, respiratory agent, sedative hypnoticagents, skin and mucous membrane agents, smoking cessation agents,steroids, sympatholytic agents, urinary tract agents, uterine relaxants,vaginal agents, vasodilator, anti-hypertensive, hyperthyroids,antihyperthyroids, anti-asthmatics, nucleic acids; expression vectors;and antivertigo agents. Examples of antitumor or antineoplastic agentsinclude bleomycin hydrochloride, methotrexate, actinomycin D, mitomycinC, vinblastine sulfate, vincristine sulfate, daunonibicin hydrochloride,adriamynin, neocarzinostatin, cytosine arabinoside; fluorouracil,tetrahydrofuryl-5-fluorouracil, picibanil, lentinan, levamisole,bestatin, azimexon, glycyrrhizin, poly A:U, poly ICLC and the like.

Examples of the antibiotics include gentamicin, dibekacin, kanendomycin,lividomycin, tobromycin, amikacin, fradiomycin, sisomysin, tetracycline,oxytetracycline, roliteracycline, doxycycline, ampicillin, piperacillin,ticarcillin, cefalotin, cefaloridine, cefotiam, cefsulodin,cefinenoxime, cefmetazole, cefazollin, cefataxim, cefoperazone,ceftizoxime, moxolactame, thienamycin, sulfazecine, azusleonam, saltsthereof, and the like. Examples of the sedative include chlorpromazine,prochloperazine, trifluoperazine, atropine, scopolamine, salts thereofand the like. Examples of the muscle relaxant include pridinol,tubocurarine, pancuronium and the like. Examples of the antiepilepticagent include phenytoin, ethosuximide, acetazolamide, chlordiazepoxideand the like. Examples of the antidepressant include imipramine,clomipramine, onxiptiline, phenelzine and the like. Examples of theantidiabetic agent include: glymidine, glipizide, phenformin, buformin,metformin and the like.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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1. A unit-dose delivery system used as a template for use in a drypowder inhaler comprising: a unit-dose delivery system comprising one ormore concave indentations; a cover positioned to sealed the one or moreconcave indentations; and a brittle matrix medicinal formulationappropriate for pulmonary delivery in at least one of the one or moreconcave indentations, wherein the brittle matrix medicinal formulationcomprises a non-tightly packed porous flocculated web matrix comprisingone or more brittle-matrix particles of one or more active agents,wherein a portion of the one or more brittle-matrix particles isdelivered and templated by the formation of one or more particles uponatomization from the unit-dose delivery system using a dry powderinhaler to form a respirable porous particle for deep lung delivery. 2.The medicinal formulation of claim 1, wherein the one or more activeagents comprise itraconazole, voriconazole, paclitaxel, sirolimus,cyclosporin, an inhalable medicinally active drug for treatment ofasthma, copd, or interstitial lung disease, mycophenolic acid or a saltthereof, tacrolimus and lactose, or tacrolimus.
 3. The medicinalformulation of claim 1, wherein the one or more active agents comprise alow molecular weight drug, a high molecular weight drug, a peptide, aprotein or a combination thereof.
 4. The medicinal formulation of claim1, wherein the one or more brittle-matrix particles comprise particlesin the form that enables delivery of the pharmaceutically active drug tothe lung, such as in the form of rods or plates.
 5. The medicinalformulation of claim 1, wherein the one or more brittle-matrix particlesare formed by rapid freezing.
 6. The medicinal formulation of claim 1,wherein the one or more brittle-matrix particles are formed by freezingand lyophilizing directly in the unit-dose delivery system.
 7. Themedicinal formulation of claim 1, wherein the unit-dose pack is ablister pack.
 8. The medicinal formulation of claim 1, wherein thebrittle matrix particles have skeletal densities less than about 1 g/mL,less than about 0.1 g/mL, and less than about 0.05 g/mL.
 9. Themedicinal formulation of claim 1, wherein the brittle matrix particleshave a Carr's index or measure of compressibility of greater than about10, greater than about 20, greater than about
 35. 10. A medicinalformulation for use in a dry powder inhaler comprising: a non-tightlypacked porous flocculated web composition comprising one or morebrittle-matrix particles of one or more active agents, wherein a portionof the one or more brittle-matrix particles is templated by a patientand/or device induced shearing energy to form a porous particle for deeplung delivery.
 11. The medicinal formulation of claim 10, wherein theone or more active agents comprise itraconazole, voriconazole,paclitaxel, sirolimus, cyclosporin, an inhalable medicinally active drugfor treatment of asthma, copd, or interstitial lung disease,mycophenolic acid or a salt thereof, tacrolimus and lactose, ortacrolimus.
 12. The medicinal formulation of claim 10, wherein the oneor more active agents comprise a low molecular weight drug, a highmolecular weight drug, a peptide, a protein or a combination thereof.13. The medicinal formulation of claim 10, wherein the one or moreactive agents are selected from a protein, a peptide, a vasoactivepeptide, an immunoglobulin, an immunomodulating protein, a hematopoieticfactor, insulin, an insulin analog, amylin, an antibiotic, an antibodyan antigen, an interleukin, an interferon, an erythropoietin, a heparin,a thrombolytic, an antitrypsin, an enzyme, an anti-protease, a hormone,a growth factor, a nucleic acid, an oligonucleotide, an antisense agentand mixtures thereof
 14. The medicinal formulation of claim 10, whereinthe non-tightly packed porous flocculated web composition comprises oneor more anisotropic particles with aspect ratios greater than
 1. 15. Themedicinal formulation of claim 10, wherein the one or morebrittle-matrix particles comprise a non-tightly packed porousflocculated web matrix comprising one or more brittle-matrix particlesof one or more active agents, wherein a portion of the one or morebrittle-matrix particles is delivered and templated by the formation ofone or more particles upon atomization.
 16. The medicinal formulation ofclaim 10, wherein the one or more brittle-matrix particles are formed byfreezing.
 17. The medicinal formulation of claim 10, wherein the one ormore brittle-matrix particles are formed by freezing and lyophilizingdirectly in the unit-dose delivery system.
 18. The medicinal formulationof claim 10, wherein the one or more active agents comprise natamycin,flucytosine, miconazole, fluconazole, itraconazole, clotrimazole,econazole, miconazole, ravuconazole, oxiconazole, sulconazole,terconazole, tioconazole, fenticonazole, bifonazole, oxiconazole,ketoconazole, isoconazole, tolnaftate, amorolfine, terbinafine,voriconazol, posaconazol, albumin, tacrolimus and lactose, tacrolimus orthe pharmacologically acceptable salts, metal complexes or mixturethereof
 19. A method of making a dispersible brittle templatedcomposition for a dry powder inhaler system comprising the steps of:cooling a unit-dose delivery system intended for one or more metereddoses for inhalation; depositing one or more drops of a drug solution onthe unit-dose delivery system, wherein the drug solution comprises oneor more active pharmaceutical ingredients, one or more solvents, and oneor more excipients, where said drop freezes upon contact with thepackaging material; lyophilizing the pharmaceutical product to produce anon-tightly packed brittle matrix; equilibrating the non-tightly packedbrittle matrix to room temperature; and combining the non-tightly packedbrittle matrix with a suitable dry powder inhalation device.
 20. Themethod of 19, wherein the non-tightly packed brittle matirx compositioncomprise particles in the form of a web of interconnected rods orplates.
 21. The method of 19, wherein the non-tightly packed brittlematrix composition comprise particles in the form of a nanostructuredweb.
 22. The method of claim 19, wherein the one or more active agentscomprise itraconazole, paclitaxel, steroids, asthma drugs,immunosuppressants, anti-fungal drugs, or anti-cancer drugs.
 23. Thecomposition of claim 19 wherein active agents may be combined with apharmaceutical excipient suitable for inhalation.
 24. The composition ofclaim 19, wherein the one or more brittle matrix particles ofanisotropic particles comprise a low molecular weight drug, a highmolecular weight drug, a peptide, a protein or a combination thereof.25. The method of claim 19, wherein the one or more active agents areselected from a protein, a peptide, a vasoactive peptide, animmunoglobulin, an immunomodulating protein, a hematopoietic factor,insulin, an insulin analog, amylin, an antibiotic, an antibody anantigen, an interleukin, an interferon, an erythropoietin, a heparin, athrombolytic, an antitrypsin, an enzyme, an anti-protease, a hormone, agrowth factor, a nucleic acid, an oligonucleotide, an antisense agent,albumin and mixtures thereof.
 26. A drug product produced by the processof claim
 19. 27. A method of making a dispersible brittle templatedcomposition for a dry powder inhaler system comprising the steps of:forming a non-tightly packed brittle matrix from one or more drops ofdrug solution comprising one or more active pharmaceutical ingredients,one or more solvents, and one or more excipients; lyophilizing thenon-tightly packed brittle matrix; equilibrating the non-tightly packedbrittle matrix to room temperature; portioning the non-tightly packedbrittle matrix into sample doses; and combining the non-tightly packedbrittle matrix with a suitable dry powder inhalation device.
 28. Amedicinal formulation for use in a dry powder inhaler comprising: anon-tightly packed porous flocculated web composition comprising one ormore brittle-matrix particles of one or more active agents, wherein aportion of the one or more brittle-matrix particles is templated by apatient and/or device induced shearing energy to form a porous particlefor deep lung delivery.
 29. A medicinal formulation of claim 28, whereinthe brittle-matrix powders are delivered in a unit-dose delivery system,such as a blister pack, a capsule from a single-dose or multiple-doseDPI device or a reservoir from a multi-dose DPI device.
 30. Themedicinal formulation of claim 28, wherein the one or more active agentscomprise itraconazole, paclitaxel, tacrolimus and lactose, ortacrolimus.
 31. The medicinal formulation of claim 28, wherein the oneor more active agents comprise a low molecular weight drug, a highmolecular weight drug, a peptide, a protein or a combination thereof.32. The medicinal formulation of claim 28, wherein the brittle matrixparticles have skeletal densities less than about 1 g/mL, less thanabout 0.1 g/mL, and less than about 0.05 g/mL.
 33. The medicinalformulation of claim 28, wherein the brittle matrix particles have aCarr's index (measure of compressibility) of greater than about 10,greater than about 20, greater than about
 35. 34. The medicinalformulation of claim 28, wherein the one or more active agents comprisenatamycin, flucytosine, miconazole, fluconazole, itraconazole,clotrimazole, econazole, miconazole, ravuconazole, oxiconazole,sulconazole, terconazole, tioconazole, fenticonazole, bifonazole,oxiconazole, ketoconazole, isoconazole, tolnaftate, amorolfine,terbinafine, voriconazol, posaconazol, albumin or the pharmacologicallyacceptable salts, metal complexes or mixture thereof.